Anti-IL-12 antibodies, compositions, methods and uses

ABSTRACT

The present invention relates to at least one novel anti-IL-12 antibodies, including isolated nucleic acids that encode at least one anti-IL-12 antibody, IL-12, vectors, host cells, transgenic animals or plants, and methods of making and using thereof, including therapeutic compositions, methods and devices.

[0001] This application is based in part on, and claims priority to,U.S. Provisional No. 60/223,358 filed Aug. 7, 2000 and No. 60/236,827filed Sep. 29, 2000, each of which is entirely incorporated herein byreference.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates to antibodies, including specifiedportions or variants, specific for at least one Interleukin-12 (IL-12)protein or fragment thereof, as well as nucleic acids encoding suchanti-IL-12 antibodies, complementary nucleic acids, vectors, host cells,and methods of making and using thereof, including therapeuticformulations, administration and devices.

[0004] 2. Related Art

[0005] Interleukin-12 (IL-12) is a heterodimeric cytokine consisting ofglycosylated polypeptide chains of 35 and 40 kD which are disulfidebonded. The cytokine is synthesized and secreted by antigen presentingcells including dendritic cells, monocytes, macrophages, B cells,Langerhans cells and keratinocytes as well as natural killer (NK) cells.IL-12 mediates a variety of biological processes and has been referredto as NK cell stimulatory factor (NKSF), T-cell stimulating factor,cytotoxic T-lymphocyte maturation factor and EBV-transformed B-cell linefactor (Curfs, J. H. A. J., et al., Clinical Microbiology Reviews,10:742-780 (1997)).

[0006] Interleukin-12 can bind to the IL-12 receptor expressed on theplasma membrane of cells (e.g., T cells, NK cell), thereby altering(e.g., initiating, preventing) biological processes. For example, thebinding of IL-12 to the IL-12 receptor can stimulate the proliferationof pre-activated T cells and NK cells, enhance the cytolytic activity ofcytotoxic T cells (CTL), NK cells and LAK (lymphokine activated killer)cells, induce production of gamma interferon (IFN GAMMA) by T cells andNK cells and induce differentiation of naive Th0 cells into Th1 cellsthat produce IFN GAMMA and IL-2 (Trinchieri, G., Annual Review ofImmunology, 13:251-276 (1995)). In particular, IL-12 is vital for thegeneration of cytolytic cells (e.g., NK, CTL) and for mounting acellular immune response (e.g., a Th1 cell mediated immune response).Thus, IL-12 is critically important in the generation and regulation ofboth protective immunity (e.g., eradication of infections) andpathological immune responses (e.g., autoimmunity) (Hendrzak, J. A. andBrunda, M. J., Laboratory Investigation, 72:619-637 (1995)).Accordingly, an immune response (e.g., protective or pathogenic) can beenhanced, suppressed or prevented by manipulation of the biologicalactivity of IL-12 in vivo, for example, by means of an antibody.

[0007] Non-human mammalian, chimeric, polyclonal (e.g., anti-sera)and/or monoclonal antibodies (Mabs) and fragments (e.g., proteolyticdigestion or fusion protein products thereof) are potential therapeuticagents that are being investigated in some cases to attempt to treatcertain diseases. However, such antibodies or fragments can elicit animmune response when administered to humans. Such an immune response canresult in an immune complex-mediated clearance of the antibodies orfragments from the circulation, and make repeated administrationunsuitable for therapy, thereby reducing the therapeutic benefit to thepatient and limiting the readministration of the antibody or fragment.For example, repeated administration of antibodies or fragmentscomprising non-human portions can lead to serum sickness and/oranaphalaxis. In order to avoid these and other problems, a number ofapproaches have been taken to reduce the immunogenicity of suchantibodies and portions thereof, including chimerization andhumanization, as well known in the art. These and other approaches,however, still can result in antibodies or fragments having someimmunogenicity, low affinity, low avidity, or with problems in cellculture, scale up, production, and/or low yields. Thus, such antibodiesor fragments can be less than ideally suited for manufacture or use astherapeutic proteins.

[0008] Accordingly, there is a need to provide anti-IL-12 antibodies orfragments that overcome one more of these problems, as well asimprovements over known antibodies or fragments thereof.

SUMMARY OF THE INVENTION

[0009] The present invention provides isolated human, primate, rodent,mammalian, chimeric, humanized and/or CDR-grafted anti-IL-12 antibodies,immunoglobulins, cleavage products and other specified portions andvariants thereof, as well as anti-IL-12 antibody compositions, encodingor complementary nucleic acids, vectors, host cells, compositions,formulations, devices, transgenic animals, transgenic plants, andmethods of making and using thereof, as described and enabled herein, incombination with what is known in the art.

[0010] The present invention also provides at least one isolatedanti-IL-12 antibody as described herein. An antibody according to thepresent invention includes any protein or peptide containing moleculethat comprises at least a portion of an immunoglobulin molecule, such asbut not limited to at least one complementarity determining region (CDR)of a heavy or light chain or a ligand binding portion thereof, a heavychain or light chain variable region, a heavy chain or light chainconstant region, a framework region, or any portion thereof, that can beincorporated into an antibody of the present invention. An antibody ofthe invention can include or be derived from any mammal, such as but notlimited to a human, a mouse, a rabbit, a rat, a rodent, a primate, orany combination thereof, and the like.

[0011] The present invention provides, in one aspect, isolated nucleicacid molecules comprising, complementary, or hybridizing to, apolynucleotide encoding specific anti-IL-12 antibodies, comprising atleast one specified sequence, domain, portion or variant thereof. Thepresent invention further provides recombinant vectors comprising saidanti-IL-12 antibody nucleic acid molecules, host cells containing suchnucleic acids and/or recombinant vectors, as well as methods of makingand/or using such antibody nucleic acids, vectors and/or host cells.

[0012] At least one antibody of the invention binds at least onespecified epitope specific to at least one IL-12 protein, subunit,fragment, portion or any combination thereof. The at least one epitopecan comprise at least one antibody binding region that comprises atleast one portion of said protein, which epitope is preferably comprisedof at least 1-5 amino acids of at least one portion thereof, such as butnot limited to, at least one functional, extracellular, soluble,hydrophillic, external or cytoplasmic domain of said protein, or anyportion thereof.

[0013] The at least one antibody can optionally comprise at least onespecified portion of at least one complementarity determining region(CDR) (e.g., CDR1, CDR2 or CDR3 of the heavy or light chain variableregion) and/or at least one constant or variable framework region or anyportion thereof. The at least one antibody amino acid sequence canfurther optionally comprise at least one specified substitution,insertion or deletion as described herein or as known in the art.

[0014] The present invention also provides at least one isolatedanti-IL-12 antibody as described herein, wherein the antibody has atleast one activity, such as, but not limited to: (i) inhibition of IL-12induced IFN-gamma secretion; (ii) inhibition of LAK cell cytotoxicity;(iii) inhibition of IFN gamma mRNA transription; (iv) inhibition ofintracellular IFN gamma CD3+ cells; and/or (v) CD95 expression. See,e.g., Chan, et al., (1992). J. Immunol. 148(1): 92-98; Chan, et al.,(1991). J. Exp. Med. 173(4): 869-79; Chehimi, et al., (1992) J. Exp.Med. 175(3): 789-96; Medvedev, et al., (1997) Cytokine 9(6): 394-404.A(n) anti-IL-12 antibody can thus be screened for a correspondingactivity according to known methods, such as but not limited to at leastone biological activity towards a IL-12 protein.

[0015] The present invention further provides at least one IL-12anti-idiotype antibody to at least one IL-12 antibody of the presentinvention. The anti-idiotype antibody includes any protein or peptidecontaining molecule that comprises at least a portion of animmunoglobulin molecule, such as but not limited to at least onecomplementarity determining region (CDR) of a heavy or light chain or aligand binding portion thereof, a heavy chain or light chain variableregion, a heavy chain or light chain constant region, a frameworkregion, or any portion thereof, that can be incorporated into anantibody of the present invention. An antibody of the invention caninclude or be derived from any mammal, such as but not limited to ahuman, a mouse, a rabbit, a rat, a rodent, a primate, and the like.

[0016] The present invention provides, in one aspect, isolated nucleicacid molecules comprising, complementary, or hybridizing to, apolynucleotide encoding at least one IL-12 anti-idiotype antibody,comprising at least one specified sequence, domain, portion or variantthereof. The present invention further provides recombinant vectorscomprising said IL-12 anti-idiotype antibody encoding nucleic acidmolecules, host cells containing such nucleic acids and/or recombinantvectors, as well as methods of making and/or using such anti-idiotypeantibody nucleic acids, vectors and/or host cells.

[0017] The present invention also provides at least one method forexpressing at least one anti-IL-12 antibody, or IL-12 anti-idiotypeantibody, in a host cell, comprising culturing a host cell as describedherein under conditions wherein at least one anti-IL-12 antibody isexpressed in detectable and/or recoverable amounts.

[0018] The present invention also provides at least one compositioncomprising (a) an isolated anti-IL-12 antibody encoding nucleic acidand/or antibody as described herein; and (b) a suitable carrier ordiluent. The carrier or diluent can optionally be pharmaceuticallyacceptable, according to known carriers or diluents. The composition canoptionally further comprise at least one further compound, protein orcomposition.

[0019] The present invention further provides at least one anti-IL-12antibody method or composition, for administering a therapeuticallyeffective amount to modulate or treat at least one IL-12 relatedcondition in a cell, tissue, organ, animal or patient and/or, prior to,subsequent to, or during a related condition, as described herein.

[0020] The present invention also provides at least one composition,device and/or method of delivery of a therapeutically orprophylactically effective amount of at least one anti-IL-12 antibody,according to the present invention.

[0021] The present invention further provides at least one anti-IL-12antibody method or composition, for diagnosing at least one IL-12related condition in a cell, tissue, organ, animal or patient and/or,prior to, subsequent to, or during a related condition, as describedherein.

[0022] The present invention also provides at least one composition,device and/or method of delivery for diagnosing of at least oneanti-IL-12 antibody, according to the present invention.

DESCRIPTION OF THE FIGURES

[0023]FIGS. 1A and 1B are graphs showing concentration-dependent bindingof human anti-IL-12 mAbs to immobilized human IL-12. Anti-IL-12antibodies were serially diluted in 1% BSA/PBS and incubated on rhIL-12coated plates for 1 hour at 37° C. Plates were washed twice with 0.02%Tween 20 (polyoxyethylene (20) sorbitan monolaurate), 0.15M saline andthen probed with horse radish peroxidase (HRP) labeled goat anti-humanIgG kappa specific antibody for 1 hour at room temperature. Plates wereagain washed, developed with o-phenylenediamine (OPD) substrate and theoptical density (OD) of each well was measured at 490 nm.

[0024]FIG. 2: Lanes from left to right in Figures A and B contain humanIL-12, human IL-12 p40, murine IL-12, and prestained molecular weightmarkers. FIG. 2A shows bands stained from total protein. The primarybands in each lane are human IL-12 (75 kd), p40 human IL-12 (40 kd), andmurine IL-12 (75 kd). FIG. 2B shows a western blot prepared from a gelidentical to that shown in FIG. 2A. Blot was reacted with C340 followedby HRP labeled goat anti-human IgG and specifically detected human IL-12(monomer and multimers) and human IL-12 p40 only. A control blot (notshown) reacted with HRP labeled goat anti-human IgG did not display anybands.

[0025]FIG. 3: Reverse transcription-PCR analysis of IFNγ gene expressionin human PBL's treated with IL-2, IL-12, IL-2+IL-12 with and withoutanti-IL-12 antibody C340, 8.6.2, isotype control antibody. Total RNA wasreverse transcribed, amplified by PCR using gene-specific primers. Thelevel of β-actin mRNA in each sample was also determined which served asa control for mRNA integrity and content.

[0026]FIG. 4 is a histogram showing that human anti-IL-12 mAb (C340)inhibits production of interferon-y (IFNγ) by monocyte depleted CD3+peripheral blood mononuclear cells (PBMC) stimulated with IL-2 plusIL-12. PBMC were cultured for five hours in control media (no addedcytokines), media supplemented with IL-12 (0.1 ng/ml) plus IL-2 (50IU/ml) (IL-12/IL-2), control media containing mAb C340 (10 μg/ml) andIL-12/IL-2 media containing mAb C340 (10 μg/ml). Intracellular IFNγ wasmeasured by two color immunostaining with CD3-PE and IFNγ-FITC. Data areshown for one donor.

[0027]FIG. 5 is a graph showing dose-dependent inhibition of IFNγsecretion by IL-2 plus IL-12 stimulated peripheral blood lymphocyteswith two different lots of a human anti-IL-12 mAb (C340). Human PBL(8×106/ml) were cultured for 24 hours with 10 U/ml IL-2, IL-2 plus 400pg/ml IL-12, or IL-2 plus IL-12 and mAb C340 as indicated. The culturesupernatents were removed and assayed for IFNγ by EIA.

[0028]FIG. 6 is a histogram showing dose-dependent inhibition of IL-12plus IL-2 induced LAK cell cytotoxicity by a human anti-IL-12 mAb(C340). LAK effector cells (human PBL, 8×106/ml) were cultured for 24hours with IL-12 (400 pg/ml) plus IL-2 (10 U/ml) and mAb C340 (5000ng/ml or 50 ng/ml as indicated). The LAK effector cells were washed andcultured with 51Cr labeled Raji target cells for four hours at aneffector to target (E:T) ration of 80:1, and the quantity of 51Crreleased into the media upon Raji cell lysis was measured. Results areexpressed as the mean of three normal donors standard error. IL-12positive control (IL-12) is effector cells incubated with IL-12 andwithout antibody. Background (BKGD) is effector cells incubated withoutIL-12 or antibody.

[0029]FIGS. 7A and 7B are histograms showing that IL-12 plusIL-2-induced expression of CD95 on CD3+ peripheral blood mononuclearcells is inhibited by human anti-IL-12 mAb (C340). PBMC were culturedfor 72 hours in media containing 0.1 ng/ml IL-12 and a suboptimal doseof IL-2 (50 IU/ml) in the presence or absence of mAb C340 (10 μg/ml).CD95 expression was measured flow cytometry of cells stained withanti-CD95-FITC. Gating was performed using two-color analysis (CD3 orCD56-PE vs. CD95-FITC) and forward vs. orthogonal light scatter.

[0030]FIG. 8 is a graph showing that recombinant human anti-human IL-12antibodies (rC340) bind to immobilized IL-12 in a manner that isindistinguishable from purified mAb C340. The concentration of rC340 inthe supernatants of three rC340-producing recombinant cell lines wasdetermined, and the supernatants were evaluated for IL-12 binding in anELISA. Plates were coated with 2 μg/ml human IL-12 and incubated withpurified mAb C340 from the original hybridoma (standard) or thesupernatants of recombinant cell lines. IL-12-bound antibody wasdetected using alkaline phosphatase-conjugated goat anti-human IgG(heavy chain+light chain).

[0031] FIGS. 9A-9C are graphs showing growth kinetics and the quantityof antibody secreted by three independently-derived rC340-producingrecombinant cell subclones (FIG. 9A, subclone C379B; FIG. 9B, subcloneC381A; FIG. 9C, subclone C389A). Recombinant cells were seeded into T75flasks at a starting density of 2×10⁵ cells/ml in standard media. Atvarious times, cells were resuspended and the number of live cells andthe quantity (μg/ml) of rC340 in the media were determined.

DESCRIPTION OF THE INVENTION

[0032] The present invention provides isolated, recombinant and/orsynthetic anti-IL-12 human, primate, rodent, mammalian, chimeric,humanized or CDR-grafted, antibodies and IL-12 anti-idiotype antibodiesthereto, as well as compositions and encoding nucleic acid moleculescomprising at least one polynucleotide encoding at least one anti-IL-12antibody or anti-idiotype antibody. The present invention furtherincludes, but is not limited to, methods of making and using suchnucleic acids and antibodies and anti-idiotype antibodies, includingdiagnostic and therapeutic compositions, methods and devices.

[0033] As used herein, an “anti-Interleukin-12 antibody,” “anti-IL-12antibody,” “anti-IL-12 antibody portion,” or “anti-IL-12 antibodyfragment” and/or “anti-IL-12 antibody variant” and the like include anyprotein or peptide containing molecule that comprises at least a portionof an immunoglobulin molecule, such as but not limited to at least onecomplementarity determinng region (CDR) of a heavy or light chain or aligand binding portion thereof, a heavy chain or light chain variableregion, a heavy chain or light chain constant region, a frameworkregion, or any portion thereof, or at least one portion of an IL-12receptor or binding protein, which can be incorporated into an antibodyof the present invention. Such antibody optionally further affects aspecific ligand, such as but not limited to where such antibodymodulates, decreases, increases, antagonizes, angonizes, mitigates,aleviates, blocks, inhibits, abrogates and/or interferes with at leastone IL-12 activity or binding, or with IL-12 receptor activity orbinding, in vitro, in situ and/or in vivo. As a non-limiting example, asuitable anti-IL-12 antibody, specified portion or variant of thepresent invention can bind at least one IL-12, or specified portions,variants or domains thereof. A suitable anti-IL-12 antibody, specifiedportion, or variant can also optionally affect at least one of IL-12activity or function, such as but not limited to, RNA, DNA or proteinsynthesis, IL-12 release, IL-12 receptor signaling, membrane IL-12cleavage, IL-12 activity, IL-12 production and/or synthesis. The term“antibody” is further intended to encompass antibodies, digestionfragments, specified portions and variants thereof, including antibodymimetics or comprising portions of antibodies that mimic the structureand/or function of an anitbody or specified fragment or portion thereof,including single chain antibodies and fragments thereof. Functionalfragments include antigen-binding fragments that bind to a mammalianIL-12. For example, antibody fragments capable of binding to IL-12 orportions thereof, including, but not limited to Fab (e.g., by papaindigestion), Fab′ (e.g., by pepsin digestion and partial reduction) andF(ab′)₂ (e.g., by pepsin digestion), facb (e.g., by plasmin digestion),pFc′ (e.g., by pepsin or plasmin digestion), Fd (e.g., by pepsindigestion, partial reduction and reaggregation), Fv or scFv (e.g., bymolecular biology techniques) fragments, are encompassed by theinvention (see, e.g., Colligan, Immunology, supra).

[0034] Such fragments can be produced by enzymatic cleavage, syntheticor recombinant techniques, as known in the art and/or as describedherein. antibodies can also be produced in a variety of truncated formsusing antibody genes in which one or more stop codons have beenintroduced upstream of the natural stop site. For example, a combinationgene encoding a F(ab′)₂ heavy chain portion can be designed to includeDNA sequences encoding the CH, domain and/or hinge region of the heavychain. The various portions of antibodies can be joined togetherchemically by conventional techniques, or can be prepared as acontiguous protein using genetic engineering techniques.

[0035] As used herein, the term “human antibody” refers to an antibodyin which substantially every part of the protein (e.g., CDR, framework,C_(L), C_(H) domains (e.g., C_(H) 1, C_(H) 2, C_(H) 3), hinge, (V_(L),V_(H))) is substantially non-immunogenic in humans, with only minorsequence changes or variations. Similarly, antibodies designated primate(monkey, babboon, chimpanzee, etc.), rodent (mouse, rat, rabbit, guineapid, hamster, and the like) and other mammals designate such species,sub-genus, genus, sub-family, family specific antibodies. Further,chimeric antibodies include any combination of the above. Such changesor variations optionally and preferably retain or reduce theimmunogenicity in humans or other species relative to non-modifiedantibodies. Thus, a human antibody is distinct from a chimeric orhumanized antibody. It is pointed out that a human antibody can beproduced by a non-human animal or prokaryotic or eukaryotic cell that iscapable of expressing functionally rearranged human immunoglobulin(e.g., heavy chain and/or light chain) genes. Further, when a humanantibody is a single chain antibody, it can comprise a linker peptidethat is not found in native human antibodies. For example, an Fv cancomprise a linker peptide, such as two to about eight glycine or otheramino acid residues, which connects the variable region of the heavychain and the variable region of the light chain. Such linker peptidesare considered to be of human origin.

[0036] Bispecific, heterospecific, heteroconjugate or similar antibodiescan also be used that are monoclonal, preferably human or humanized,antibodies that have binding specificities for at least two differentantigens. In the present case, one of the binding specificities is forat least one IL-12 protein, the other one is for any other antigen.Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy chain-light chainpairs, where the two heavy chains have different specificities (Milsteinand Cuello, Nature 305:537 (1983)). Because of the random assortment ofimmunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of 10 different antibody molecules, of whichonly one has the correct bispecific structure. The purification of thecorrect molecule, which is usually done by affinity chromatographysteps, is rather cumbersome, and the product yields are low. Similarprocedures are disclosed, e.g., in WO 93/08829, U.S. Pat. Nos.6,210,668, 6,193,967, 6,132,992, 6,106,833, 6,060,285, 6,037,453,6,010,902, 5,989,530, 5,959,084, 5,959,083,5,932,448, 5,833,985,5,821,333, 5,807,706, 5,643,759, 5,601,819, 5,582,996, 5,496,549,4,676,980, WO 91/00360, WO 92/00373, EP 03089, Traunecker et al., EMBOJ. 10:3655 (1991), Suresh et al., Methods in Enzymology 121:210 (1986),each entirely incorporated herein by reference.

[0037] Anti-IL-12 antibodies (also termed IL-12 antibodies) useful inthe methods and compositions of the present invention can optionally becharacterized by high affinity binding to IL-12 and optionally andpreferably having low toxicity. In particular, an antibody, specifiedfragment or variant of the invention, where the individual components,such as the variable region, constant region and framework, individuallyand/or collectively, optionally and preferably possess lowimmunogenicity, is useful in the present invention. The antibodies thatcan be used in the invention are optionally characterized by theirability to treat patients for extended periods with measurablealleviation of symptoms and low and/or acceptable toxicity. Low oracceptable immunogenicity and/or high affinity, as well as othersuitable properties, can contribute to the therapeutic results achieved.“Low immunogenicity” is defined herein as raising significant HAHA, HACAor HAMA responses in less than about 75%, or preferably less than about50% of the patients treated and/or raising low titres in the patienttreated (less than about 300, preferably less than about 100 measuredwith a double antigen enzyme immunoassay) (see, e.g., Elliott et al.,Lancet 344:1125-1127 (1994), entirely incorporated herein by reference).

[0038] Utility

[0039] The isolated nucleic acids of the present invention can be usedfor production of at least one anti-IL-12 antibody or specified variantthereof, which can be used to measure or effect in an cell, tissue,organ or animal (including mammals and humans), to diagnose, monitor,modulate, treat, alleviate, help prevent the incidence of, or reduce thesymptoms of, at least one IL-12 condition, selected from, but notlimited to, at least one of an immune disorder or disease, acardiovascular disorder or disease, an infectious, malignant, and/orneurologic disorder or disease, or other known or specified IL-12related condition.

[0040] Such a method can comprise administering an effective amount of acomposition or a pharmaceutical composition comprising at least oneanti-IL-12 antibody to a cell, tissue, organ, animal or patient in needof such modulation, treatment, alleviation, prevention, or reduction insymptoms, effects or mechanisms. The effective amount can comprise anamount of about 0.001 to 500 mg/kg per single (e.g., bolus), multiple orcontinuous administration, or to achieve a serum concentration of0.01-5000 μg/ml serum concentration per single, multiple or continuousadminstration, or any effective range or value therein, as done anddetermined using known methods, as described herein or known in therelevant arts.

[0041] Citations

[0042] All publications or patents cited herein are entirelyincorporated herein by reference as they show the state of the art atthe time of the present invention and/or to provide description andenablement of the present invention. Publications refer to anyscientific or patent publications, or any other information available inany media format, including all recorded, electronic or printed formats.The following references are entirely incorporated herein by reference:Ausubel, et al., ed., Current Protocols in Molecular Biology, John Wiley& Sons, Inc., NY, N.Y. (1987-2001); Sambrook, et al., Molecular Cloning:A Laboratory Manual, 2^(nd) Edition, Cold Spring Harbor, N.Y. (1989);Harlow and Lane, antibodies, a Laboratory Manual, Cold Spring Harbor,N.Y. (1989); Colligan, et al., eds., Current Protocols in Immunology,John Wiley & Sons, Inc., NY (1994-2001); Colligan et al., CurrentProtocols in Protein Science, John Wiley & Sons, NY, N.Y., (1997-2001).

[0043] Antibodies of the Present Invention

[0044] At least one anti-IL-12 antibody of the present invention can beoptionally produced by a cell line, a mixed cell line, an immortalizedcell or clonal population of immortalized cells, as well known in theart. See, e.g., Ausubel, et al., ed., Current Protocols in MolecularBiology, John Wiley & Sons, Inc., NY, N.Y. (1987-2001); Sambrook, etal., Molecular Cloning: A Laboratory Manual, 2^(nd) Edition, Cold SpringHarbor, N.Y. (1989); Harlow and Lane, antibodies, a Laboratory Manual,Cold Spring Harbor, N.Y. (1989); Colligan, et al., eds., CurrentProtocols in Immunology, John Wiley & Sons, Inc., NY (1994-2001);Colligan et al., Current Protocols in Protein Science, John Wiley &Sons, NY, N.Y., (1997-2001), each entirely incorporated herein byreference.

[0045] Human antibodies that are specific for human IL-12 proteins orfragments thereof can be raised against an appropriate immunogenicantigen, such as isolated and/or IL-12 protein or a portion thereof(including synthetic molecules, such as synthetic peptides). Otherspecific or general mammalian antibodies can be similarly raised.Preparation of immunogenic antigens, and monoclonal antibody productioncan be performed using any suitable technique.

[0046] In one approach, a hybridoma is produced by fusing a suitableimmortal cell line (e.g., a myeloma cell line such as, but not limitedto, Sp2/0, Sp2/0-AG14, NSO, NS1, NS2, AE-1, L.5, >243, P3X63Ag8.653, Sp2SA3, Sp2 MAI, Sp2 SS1, Sp2 SA5, U937, MLA 144, ACT IV, MOLT4, DA-1,JURKAT, WEHI, K-562, COS, RAJI, NIH 3T3, HL-60, MLA 144, NAMAIWA, NEURO2A, or the like, or heteromylomas, fusion products thereof, or any cellor fusion cell derived therefrom, or any other suitable cell line asknown in the art. See, e.g., www.atcc.org, www.lifetech.com., and thelike, with antibody producing cells, such as, but not limited to,isolated or cloned spleen, peripheral blood, lymph, tonsil, or otherimmune or B cell containing cells, or any other cells expressing heavyor light chain constant or variable or framework or CDR sequences,either as endogenous or heterologous nucleic acid, as recombinant orendogenous, viral, bacterial, algal, prokaryotic, amphibian, insect,reptilian, fish, mammalian, rodent, equine, ovine, goat, sheep, primate,eukaryotic, genomic DNA, cDNA, rDNA, mitochondrial DNA or RNA,chloroplast DNA or RNA, hnRNA, mRNA, tRNA, single, double or triplestranded, hybridized, and the like or any combination thereof. See,e.g., Ausubel, supra, and Colligan, Immunology, supra, chapter 2,entirely incorporated herein by reference.

[0047] Antibody producing cells can also be obtained from the peripheralblood or, preferably the spleen or lymph nodes, of humans or othersuitable animals that have been immunized with the antigen of interest.Any other suitable host cell can also be used for expressingheterologous or endogenous nucleic acid encoding an antibody, specifiedfragment or variant thereof, of the present invention. The fused cells(hybridomas) or recombinant cells can be isolated using selectiveculture conditions or other suitable known methods, and cloned bylimiting dilution or cell sorting, or other known methods. Cells whichproduce antibodies with the desired specificity can be selected by asuitable assay (e.g., ELISA).

[0048] Other suitable methods of producing or isolating antibodies ofthe requisite specificity can be used, including, but not limited to,methods that select recombinant antibody from a peptide or proteinlibrary (e.g., but not limited to, a bacteriophage, ribosome,oligonucleotide, RNA, cDNA, or the like, display library; e.g., asavailable from Cambridge antibody Technologies, Cambridgeshire, UK;MorphoSys, Martinsreid/Planegg, DE; Biovation, Aberdeen, Scotland, UK;BioInvent, Lund, Sweden; Dyax Corp., Enzon, Affymax/Biosite; Xoma,Berkeley, Calif.; Lxsys. See, e.g., EP 368,684, PCT/GB91/01134;PCT/GB92/01755; PCT/GB92/002240; PCT/GB92/00883; PCT/GB93/00605; U.S.Ser. No. 08/350,260 (May 12, 1994); PCT/GB94/01422; PCT/GB94/02662;PCT/GB97/01835; (CAT/MRC); WO90/14443; WO90/14424; WO90/14430;PCT/US94/1234; WO92/18619; WO96/07754; (Scripps); EP 614 989(MorphoSys); WO95/16027 (Biolnvent); WO88/06630; WO90/3809 (Dyax); U.S.Pat. No. 4,704,692 (Enzon); PCT/US91/02989 (Affymax); WO89/06283; EP 371998; EP 550 400; (Xoma); EP 229 046; PCT/US91/07149 (Ixsys); orstochastically generated peptides or proteins—U.S. Pat. Nos. 5,723,323,5,763,192, 5,814,476, 5,817,483, 5,824,514, 5,976,862, WO 86/05803, EP590 689 (Ixsys, now Applied Molecular Evolution (AME), each entirelyincorporated herein by reference) or that rely upon immunization oftransgenic animals (e.g., SCID mice, Nguyen et al., Microbiol. Immunol.41:901-907 (1997); Sandhu et al., Crit. Rev. Biotechnol. 16:95-118(1996); Eren et al., Immunol. 93:154-161 (1998), each entirelyincorporated by reference as well as related patents and application)that are capable of producing a repertoire of human antibodies, as knownin the art and/or as described herein. Such techniques, include, but arenot limited to, ribosome display (Hanes et al., Proc. Natl. Acad. Sci.USA, 94:4937-4942 (May 1997); Hanes et al., Proc. Natl. Acad. Sci. USA,95:14130-14135 (November 1998)); single cell antibody producingtechnologies (e.g., selected lymphocyte antibody method (“SLAM”) (U.S.Pat. No. 5,627,052, Wen et al., J. Immunol. 17:887-892 (1987); Babcooket al., Proc. Natl. Acad. Sci. USA 93:7843-7848 (1996)); gelmicrodroplet and flow cytometry (Powell et al., Biotechnol. 8:333-337(1990); One Cell Systems, Cambridge, Mass.; Gray et al., J. Imm. Meth.182:155-163 (1995); Kenny et al., Bio/Technol. 13:787-790 (1995));B-cell selection (Steenbakkers et al., Molec. Biol. Reports 19:125-134(1994); Jonak et al., Progress Biotech, Vol. 5, In Vitro Immunization inHybridoma Technology, Borrebaeck, ed., Elsevier Science Publishers B.V., Amsterdam, Netherlands (1988)).

[0049] Methods for engineering or humanizing non-human or humanantibodies can also be used and are well known in the art. Generally, ahumanized or engineered antibody has one or more amino acid residuesfrom a source which is non-human, e.g., but not limited to mouse, rat,rabbit, non-human primate or other mammal. These human amino acidresidues are often referred to as “import” residues, which are typicallytaken from an “import” variable, constant or other domain of a knownhuman sequence. Known human Ig sequences are disclosed, e.g.,www.ncbi.nlm.nih.gov/entrez/query.fcgi; www.atcc.org/phage/hdb.html;www.sciquest.com/; www.abcam.com/;www.antibodyresource.com/onlinecomp.html;www.public.iastate.edu/˜pedro/research_tools.html;www.mgen.uni-heidelberg.de/SD/IT/IT.html;www.whfreeman.com/immunology/CH05/kuby05.htm;www.library.thinkquest.org/12429/Immune/Antibody.html;www.hhmi.org/grants/lectures/1996/vlab/;www.path.cam.ac.uk/˜mrc7/mikeimages.html; www.antibodyresource.com/;mcb.harvard.edu/BioLinks/Immunology.html.www.immunologylink.com/;pathbox.wustl.edu/˜hcenter/index.html; www.biotech.ufl.edu/˜hcl/;www.pebio.com/pa/340913/340913.html;www.nal.usda.gov/awic/pubs/antibody/;www.m.ehime-u.ac.jp/˜yasuhito/Elisa.html; www.biodesign.com/table.asp;www.icnet.uk/axp/facs/davies/links.html;www.biotech.ufl.edu/fccl/protocol.html; www.isac-net.org/sites_geo.html;aximt1.imt.uni-marburg.de/˜rek/AEPStart.html;baserv.uci.kun.nl/˜jraats/links1.html;www.recab.uni-hd.de/immuno.bme.nwu.edu/;www.mrc-cpe.cam.ac.uk/imt-doc/public/INTRO.html;www.ibt.unam.mx/vir/V_mice.html; imgt.cnusc.fr:8104/;www.biochem.ucl.ac.uk/˜martin/abs/index.html; antibody.bath.ac.uk/;abgen.cvm.tamu.edu/lab/wwwabgen.html;www.unizh.ch/˜honegger/AHOseminar/Slide01.html;www.cryst.bbk.ac.uk/˜ubcg07s/; www.mir.mrc.ac.uk/CC/ccaewg/ccaewg.htm;www.path.cam.ac.uk/˜mrc7/humanisation/TAHHP.html;www.ibt.unam.mx/vir/structure/stat_aim.html;www.biosci.missouri.edu/smithgp/index.html;www.cryst.bioc.cam.ac.uk/˜fmolina/Web-pages/Pept/spottech.html;wwwjerini.de/fr_products.htm; www.patents.ibm.com/ibm.html.Kabat et al.,Sequences of Proteins of Immunological Interest, U.S. Dept. Health(1983), each entirely incorporated herein by reference. Such importedsequences can be used to reduce immunogenicity or reduce, enhance ormodify binding, affinity, on-rate, off-rate, avidity, specificity,half-life, or any other suitable characteristic, as known in the art.Generally part or all of the non-human or human CDR sequences aremaintained while the non-human sequences of the variable and constantregions are replaced with human or other amino acids. antibodies canalso optionally be humanized with retention of high affinity for theantigen and other favorable biological properties. To achieve this goal,humanized antibodies can be optionally prepared by a process of analysisof the parental sequences and various conceptual humanized productsusing three-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the consensus and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the CDR residues aredirectly and most substantially involved in influencing antigen binding.Humanization or engineering of antibodies of the present invention canbe performed using any known method, such as but not limited to thosedescribed in, Winter (Jones et al., Nature 321:522 (1986); Riechmann etal., Nature 332:323 (1988); Verhoeyen et al., Science 239:1534 (1988)),Sims et al., J. Immunol. 151: 2296 (1993); Chothia and Lesk, J. Mol.Biol. 196:901 (1987), Carter et al., Proc. Natl. Acad. Sci. U.S.A.89:4285 (1992); Presta et al., J. Immunol. 151:2623 (1993), U.S. Pat.Nos. 5,723,323, 5,976,862, 5,824,514, 5,817,483, 5,814,476, 5,763,192,5,723,323, 5,766,886, 5,714,352, 6,204,023, 6,180,370, 5,693,762,5,530,101, 5,585,089, 5,225,539; 4,816,567, PCT/: US98/16280,US96/18978, US91/09630, US91/05939, US94/01234, GB89/01334, GB91/01134,GB92/01755; WO90/14443, WO90/14424, WO90/14430, EP 229246, each entirelyincorporated herein by reference, included references cited therein.

[0050] The anti-IL-12 antibody can also be optionally generated byimmunization of a transgenic animal (e.g., mouse, rat, hamster,non-human primate, and the like) capable of producing a repertoire ofhuman antibodies, as described herein and/or as known in the art. Cellsthat produce a human anti-IL-12 antibody can be isolated from suchanimals and immortalized using suitable methods, such as the methodsdescribed herein.

[0051] Transgenic mice that can produce a repertoire of human antibodiesthat bind to human antigens can be produced by known methods (e.g., butnot limited to, U.S. Pat. Nos. 5,770,428, 5,569,825, 5,545,806,5,625,126, 5,625,825, 5,633,425, 5,661,016 and 5,789,650 issued toLonberg et al.; Jakobovits et al. WO 98/50433, Jakobovits et al. WO98/24893, Lonberg et al. WO 98/24884, Lonberg et al. WO 97/13852,Lonberg et al. WO 94/25585, Kucherlapate et al. WO 96/34096,Kucherlapate et al. EP 0463 151 B1, Kucherlapate et al. EP 0710 719 A1,Surani et al. U.S. Pat. No. 5,545,807, Bruggemann et al. WO 90/04036,Bruggemann et al. EP 0438 474 B1, Lonberg et al. EP 0814 259 A2, Lonberget al. GB 2 272 440 A, Lonberg et al. Nature 368:856-859 (1994), Tayloret al., Int. Immunol. 6(4)579-591 (1994), Green et al, Nature Genetics7:13-21 (1994), Mendez et al., Nature Genetics 15:146-156 (1997), Tayloret al., Nucleic Acids Research 20(23):6287-6295 (1992), Tuaillon et al.,Proc Natl Acad Sci USA 90(8)3720-3724 (1993), Lonberg et al., Int RevImmunol 13(1):65-93 (1995) and Fishwald et al., Nat Biotechnol14(7):845-851 (1996), which are each entirely incorporated herein byreference). Generally, these mice comprise at least one transgenecomprising DNA from at least one human immunoglobulin locus that isfunctionally rearranged, or which can undergo functional rearrangement.The endogenous immunoglobulin loci in such mice can be disrupted ordeleted to eliminate the capacity of the animal to produce antibodiesencoded by endogenous genes.

[0052] Screening antibodies for specific binding to similar proteins orfragments can be conveniently achieved using peptide display libraries.This method involves the screening of large collections of peptides forindividual members having the desired function or structure. antibodyscreening of peptide display libraries is well known in the art. Thedisplayed peptide sequences can be from 3 to 5000 or more amino acids inlength, frequently from 5-100 amino acids long, and often from about 8to 25 amino acids long. In addition to direct chemical synthetic methodsfor generating peptide libraries, several recombinant DNA methods havebeen described. One type involves the display of a peptide sequence onthe surface of a bacteriophage or cell. Each bacteriophage or cellcontains the nucleotide sequence encoding the particular displayedpeptide sequence. Such methods are described in PCT Patent PublicationNos. 91/17271, 91/18980, 91/19818, and 93/08278. Other systems forgenerating libraries of peptides have aspects of both in vitro chemicalsynthesis and recombinant methods. See, PCT Patent Publication Nos.92/05258, 92/14843, and 96/19256. See also, U.S. Pat. Nos. 5,658,754;and 5,643,768. Peptide display libraries, vector, and screening kits arecommercially available from such suppliers as Invitrogen (Carlsbad,Calif.), and Cambridge Antibody Technologies (Cambridgeshire, UK). See,e.g., U.S. Pat. Nos. 4,704,692, 4,939,666, 4,946,778, 5,260,203,5,455,030, 5,518,889, 5,534,621, 5,656,730, 5,763,733, 5,767,260,5,856,456, assigned to Enzon; U.S. Pat. Nos. 5,223,409, 5,403,484,5,571,698, 5,837,500, assigned to Dyax, U.S. Pat. Nos. 5,427,908,5,580,717, assigned to Affymax; U.S. Pat. No. 5,885,793, assigned toCambridge antibody Technologies; U.S. Pat. No. 5,750,373, assigned toGenentech, U.S. Pat. Nos. 5,618,920, 5,595,898, 5,576,195, 5,698,435,5,693,493, 5,698,417, assigned to Xoma, Colligan, supra; Ausubel, supra;or Sambrook, supra, each of the above patents and publications entirelyincorporated herein by reference.

[0053] Antibodies of the present invention can also be prepared using atleast one anti-IL-12 antibody encoding nucleic acid to providetransgenic animals or mammals, such as goats, cows, horses, sheep, andthe like, that produce such antibodies in their milk. Such animals canbe provided using known methods. See, e.g., but not limited to, U.S.Pat. Nos. 5,827,690; 5,849,992; 4,873,316; 5,849,992; 5,994,616;5,565,362; 5,304,489, and the like, each of which is entirelyincorporated herein by reference.

[0054] Antibodies of the present invention can additionally be preparedusing at least one anti-IL-12 antibody encoding nucleic acid to providetransgenic plants and cultured plant cells (e.g., but not limited totobacco and maize) that produce such antibodies, specified portions orvariants in the plant parts or in cells cultured therefrom. As anon-limiting example, transgenic tobacco leaves expressing recombinantproteins have been successfully used to provide large amounts ofrecombinant proteins, e.g., using an inducible promoter. See, e.g.,Cramer et al., Curr. Top. Microbol. Immunol. 240:95-118 (1999) andreferences cited therein. Also, transgenic maize have been used toexpress mammalian proteins at commercial production levels, withbiological activities equivalent to those produced in other recombinantsystems or purified from natural sources. See, e.g., Hood et al., Adv.Exp. Med. Biol. 464:127-147 (1999) and references cited therein.antibodies have also been produced in large amounts from transgenicplant seeds including antibody fragments, such as single chainantibodies (scFv's), including tobacco seeds and potato tubers. See,e.g., Conrad et al., Plant Mol. Biol. 38:101-109 (1998) and referencecited therein. Thus, antibodies of the present invention can also beproduced using transgenic plants, according to know methods. See also,e.g., Fischer et al., Biotechnol. Appl. Biochem. 30:99-108 (October1999), Ma et al., Trends Biotechnol. 13:522-7 (1995); Ma et al., PlantPhysiol. 109:341-6 (1995); Whitelam et al., Biochem. Soc. Trans.22:940-944 (1994); and references cited therein. See, also generally forplant expression of antibodies, but not limited to, Each of the abovereferences is entirely incorporated herein by reference.

[0055] The antibodies of the invention can bind human IL-12 with a widerange of affinities (K_(D)). In a preferred embodiment, at least onehuman mAb of the present invention can optionally bind human IL-12 withhigh affinity. For example, a human mAb can bind human IL-12 with aK_(D) equal to or less than about 10⁻⁷ M, such as but not limited to,0.1-9.9 (or any range or value therein) X 10⁻⁷, 10⁻⁸, 10⁻⁹, 10⁻¹⁰,10⁻¹¹, 10⁻¹², 10⁻¹³ or any range or value therein.

[0056] The affinity or avidity of an antibody for an antigen can bedetermined experimentally using any suitable method. (See, for example,Berzofsky, et al., “Antibody-Antigen Interactions,” In FundamentalImmunology, Paul, W. E., Ed., Raven Press: New York, N.Y. (1984); Kuby,Janis Immunology, W. H. Freeman and Company: New York, N.Y. (1992); andmethods described herein). The measured affinity of a particularantibody-antigen interaction can vary if measured under differentconditions (e.g., salt concentration, pH). Thus, measurements ofaffinity and other antigen-binding parameters (e.g., K_(D), K_(a),K_(d)) are preferably made with standardized solutions of antibody andantigen, and a standardized buffer, such as the buffer described herein.

[0057] Nucleic Acid Molecules

[0058] Using the information provided herein, such as the nucleotidesequences encoding at least 70-100% of the contiguous amino acids of atleast one of SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8, specified fragments,variants or consensus sequences thereof, or a deposited vectorcomprising at least one of these sequences, a nucleic acid molecule ofthe present invention encoding at least one anti-IL-12 antibody can beobtained using methods described herein or as known in the art.

[0059] Nucleic acid molecules of the present invention can be in theform of RNA, such as mRNA, hnRNA, tRNA or any other form, or in the formof DNA, including, but not limited to, cDNA and genomic DNA obtained bycloning or produced synthetically, or any combinations thereof. The DNAcan be triple-stranded, double-stranded or single-stranded, or anycombination thereof. Any portion of at least one strand of the DNA orRNA can be the coding strand, also known as the sense strand, or it canbe the non-coding strand, also referred to as the anti-sense strand.

[0060] Isolated nucleic acid molecules of the present invention caninclude nucleic acid molecules comprising an open reading frame (ORF),optionally with one or more introns, e.g., but not limited to, at leastone specified portion of at least one CDR, as CDR1, CDR2 and/or CDR3 ofat least one heavy chain (e.g., SEQ ID NOS:1-3) or light chain (e.g.,SEQ ID NOS: 4-6); nucleic acid molecules comprising the coding sequencefor an anti-IL-12 antibody or variable region (e.g., SEQ ID NOS:7, 8);and nucleic acid molecules which comprise a nucleotide sequencesubstantially different from those described above but which, due to thedegeneracy of the genetic code, still encode at least one anti-IL-12antibody as described herein and/or as known in the art. Of course, thegenetic code is well known in the art. Thus, it would be routine for oneskilled in the art to generate such degenerate nucleic acid variantsthat code for specific anti-IL-12 antibodies of the present invention.See, e.g., Ausubel, et al., supra, and such nucleic acid variants areincluded in the present invention. Non-limiting examples of isolatednucleic acid molecules of the present inveniton include SEQ ID NOS:10-15, corresponding to non-limiting examples of a nucleic acidencoding, respectively, HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, LCCDR3, HC variable region and LC variable region.

[0061] In another aspect, the invention provides isolated nucleic acidmolecules encoding a(n) anti-IL-12 antibody having an amino acidsequence as encoded by the nucleic acid contained in the plasmiddeposited as designated clone names ______ and ATCC Deposit Nos. ______,respectively, deposited on ______.

[0062] As indicated herein, nucleic acid molecules of the presentinvention which comprise a nucleic acid encoding an anti-IL-12 antibodycan include, but are not limited to, those encoding the amino acidsequence of an antibody fragment, by itself; the coding sequence for theentire antibody or a portion thereof; the coding sequence for anantibody, fragment or portion, as well as additional sequences, such asthe coding sequence of at least one signal leader or fusion peptide,with or without the aforementioned additional coding sequences, such asat least one intron, together with additional, non-coding sequences,including but not limited to, non-coding 5′ and 3′ sequences, such asthe transcribed, non-translated sequences that play a role intranscription, mRNA processing, including splicing and polyadenylationsignals (for example—ribosome binding and stability of mRNA); anadditional coding sequence that codes for additional amino acids, suchas those that provide additional functionalities. Thus, the sequenceencoding an antibody can be fused to a marker sequence, such as asequence encoding a peptide that facilitates purification of the fusedantibody comprising an antibody fragment or portion.

[0063] Polynucleotides Which Selectively Hybridize to a Polynucleotideas Described Herein

[0064] The present invention provides isolated nucleic acids thathybridize under selective hybridization conditions to a polynucleotidedisclosed herein. Thus, the polynucleotides of this embodiment can beused for isolating, detecting, and/or quantifying nucleic acidscomprising such polynucleotides. For example, polynucleotides of thepresent invention can be used to identify, isolate, or amplify partialor full-length clones in a deposited library. In some embodiments, thepolynucleotides are genomic or cDNA sequences isolated, or otherwisecomplementary to, a cDNA from a human or mammalian nucleic acid library.

[0065] Preferably, the cDNA library comprises at least 80% full-lengthsequences, preferably at least 85% or 90% full-length sequences, andmore preferably at least 95% full-length sequences. The cDNA librariescan be normalized to increase the representation of rare sequences. Lowor moderate stringency hybridization conditions are typically, but notexclusively, employed with sequences having a reduced sequence identityrelative to complementary sequences. Moderate and high stringencyconditions can optionally be employed for sequences of greater identity.Low stringency conditions allow selective hybridization of sequenceshaving about 70% sequence identity and can be employed to identifyorthologous or paralogous sequences.

[0066] Optionally, polynucleotides of this invention will encode atleast a portion of an antibody encoded by the polynucleotides describedherein. The polynucleotides of this invention embrace nucleic acidsequences that can be employed for selective hybridization to apolynucleotide encoding an antibody of the present invention. See, e.g.,Ausubel, supra; Colligan, supra, each entirely incorporated herein byreference.

[0067] Construction of Nucleic Acids

[0068] The isolated nucleic acids of the present invention can be madeusing (a) recombinant methods, (b) synthetic techniques, (c)purification techniques, or combinations thereof, as well-known in theart.

[0069] The nucleic acids can conveniently comprise sequences in additionto a polynucleotide of the present invention. For example, amulti-cloning site comprising one or more endonuclease restriction sitescan be inserted into the nucleic acid to aid in isolation of thepolynucleotide. Also, translatable sequences can be inserted to aid inthe isolation of the translated polynucleotide of the present invention.For example, a hexa-histidine marker sequence provides a convenientmeans to purify the proteins of the present invention. The nucleic acidof the present invention—excluding the coding sequence—is optionally avector, adapter, or linker for cloning and/or expression of apolynucleotide of the present invention.

[0070] Additional sequences can be added to such cloning and/orexpression sequences to optimize their function in cloning and/orexpression, to aid in isolation of the polynucleotide, or to improve theintroduction of the polynucleotide into a cell. Use of cloning vectors,expression vectors, adapters, and linkers is well known in the art.(See, e.g., Ausubel, supra; or Sambrook, supra)

[0071] Recombinant Methods for Constructing Nucleic Acids

[0072] The isolated nucleic acid compositions of this invention, such asRNA, cDNA, genomic DNA, or any combination thereof, can be obtained frombiological sources using any number of cloning methodologies known tothose of skill in the art. In some embodiments, oligonucleotide probesthat selectively hybridize, under stringent conditions, to thepolynucleotides of the present invention are used to identify thedesired sequence in a cDNA or genomic DNA library. The isolation of RNA,and construction of cDNA and genomic libraries, is well known to thoseof ordinary skill in the art. (See, e.g., Ausubel, supra; or Sambrook,supra)

[0073] Nucleic Acid Screening and Isolation Methods

[0074] A cDNA or genomic library can be screened using a probe basedupon the sequence of a polynucleotide of the present invention, such asthose disclosed herein. Probes can be used to hybridize with genomic DNAor cDNA sequences to isolate homologous genes in the same or differentorganisms. Those of skill in the art will appreciate that variousdegrees of stringency of hybridization can be employed in the assay; andeither the hybridization or the wash medium can be stringent. As theconditions for hybridization become more stringent, there must be agreater degree of complementarity between the probe and the target forduplex formation to occur. The degree of stringency can be controlled byone or more of temperature, ionic strength, pH and the presence of apartially denaturing solvent such as formamide. For example, thestringency of hybridization is conveniently varied by changing thepolarity of the reactant solution through, for example, manipulation ofthe concentration of formamide within the range of 0% to 50%. The degreeof complementarity (sequence identity) required for detectable bindingwill vary in accordance with the stringency of the hybridization mediumand/or wash medium. The degree of complementarity will optimally be100%, or 70-100%, or any range or value therein. However, it should beunderstood that minor sequence variations in the probes and primers canbe compensated for by reducing the stringency of the hybridizationand/or wash medium.

[0075] Methods of amplification of RNA or DNA are well known in the artand can be used according to the present invention without undueexperimentation, based on the teaching and guidance presented herein.

[0076] Known methods of DNA or RNA amplification include, but are notlimited to, polymerase chain reaction (PCR) and related amplificationprocesses (see, e.g., U.S. Pat. Nos. 4,683,195, 4,683,202, 4,800,159,4,965,188, to Mullis, et al.; U.S. Pat. Nos. 4,795,699 and 4,921,794 toTabor, et al; U.S. Pat. No. 5,142,033 to Innis; U.S. Pat. No. 5,122,464to Wilson, et al.; U.S. Pat. No. 5,091,310 to Innis; U.S. Pat. No.5,066,584 to Gyllensten, et al; U.S. Pat. No. 4,889,818 to Gelfand, etal; U.S. Pat. No. 4,994,370 to Silver, et al; U.S. Pat. No. 4,766,067 toBiswas; U.S. Pat. No. 4,656,134 to Ringold) and RNA mediatedamplification that uses anti-sense RNA to the target sequence as atemplate for double-stranded DNA synthesis (U.S. Pat. No. 5,130,238 toMalek, et al, with the tradename NASBA), the entire contents of whichreferences are incorporated herein by reference. (See, e.g., Ausubel,supra; or Sambrook, supra.)

[0077] For instance, polymerase chain reaction (PCR) technology can beused to amplify the sequences of polynucleotides of the presentinvention and related genes directly from genomic DNA or cDNA libraries.PCR and other in vitro amplification methods can also be useful, forexample, to clone nucleic acid sequences that code for proteins to beexpressed, to make nucleic acids to use as probes for detecting thepresence of the desired mRNA in samples, for nucleic acid sequencing, orfor other purposes. Examples of techniques sufficient to direct personsof skill through in vitro amplification methods are found in Berger,supra, Sambrook, supra, and Ausubel, supra, as well as Mullis, et al.,U.S. Pat. No. 4,683,202 (1987); and Innis, et al., PCR Protocols A Guideto Methods and Applications, Eds., Academic Press Inc., San Diego,Calif. (1990). Commercially available kits for genomic PCR amplificationare known in the art. See, e.g., Advantage-GC Genomic PCR Kit(Clontech). Additionally, e.g., the T4 gene 32 protein (BoehringerMannheim) can be used to improve yield of long PCR products.

[0078] Synthetic Methods for Constructing Nucleic Acids

[0079] The isolated nucleic acids of the present invention can also beprepared by direct chemical synthesis by known methods (see, e.g.,Ausubel, et al., supra). Chemical synthesis generally produces asingle-stranded oligonucleotide, which can be converted intodouble-stranded DNA by hybridization with a complementary sequence, orby polymerization with a DNA polymerase using the single strand as atemplate. One of skill in the art will recognize that while chemicalsynthesis of DNA can be limited to sequences of about 100 or more bases,longer sequences can be obtained by the ligation of shorter sequences.

[0080] Recombinant Expression Cassettes

[0081] The present invention further provides recombinant expressioncassettes comprising a nucleic acid of the present invention. A nucleicacid sequence of the present invention, for example a cDNA or a genomicsequence encoding an antibody of the present invention, can be used toconstruct a recombinant expression cassette that can be introduced intoat least one desired host cell. A recombinant expression cassette willtypically comprise a polynucleotide of the present invention operablylinked to transcriptional initiation regulatory sequences that willdirect the transcription of the polynucleotide in the intended hostcell. Both heterologous and non-heterologous (i.e., endogenous)promoters can be employed to direct expression of the nucleic acids ofthe present invention.

[0082] In some embodiments, isolated nucleic acids that serve aspromoter, enhancer, or other elements can be introduced in theappropriate position (upstream, downstream or in intron) of anon-heterologous form of a polynucleotide of the present invention so asto up or down regulate expression of a polynucleotide of the presentinvention. For example, endogenous promoters can be altered in vivo orin vitro by mutation, deletion and/or substitution.

[0083] Vectors and Host Cells

[0084] The present invention also relates to vectors that includeisolated nucleic acid molecules of the present invention, host cellsthat are genetically engineered with the recombinant vectors, and theproduction of at least one anti-IL-12 antibody by recombinanttechniques, as is well known in the art. See, e.g., Sambrook, et al.,supra; Ausubel, et al., supra, each entirely incorporated herein byreference.

[0085] The polynucleotides can optionally be joined to a vectorcontaining a selectable marker for propagation in a host. Generally, aplasmid vector is introduced in a precipitate, such as a calciumphosphate precipitate, or in a complex with a charged lipid. If thevector is a virus, it can be packaged in vitro using an appropriatepackaging cell line and then transduced into host cells.

[0086] The DNA insert should be operatively linked to an appropriatepromoter. The expression constructs will further contain sites fortranscription initiation, termination and, in the transcribed region, aribosome binding site for translation. The coding portion of the maturetranscripts expressed by the constructs will preferably include atranslation initiating at the beginning and a termination codon (e.g.,UAA, UGA or UAG) appropriately positioned at the end of the mRNA to betranslated, with UAA and UAG preferred for mammalian or eukaryotic cellexpression.

[0087] Expression vectors will preferably but optionally include atleast one selectable marker. Such markers include, e.g., but not limitedto, methotrexate (MTX), dihydrofolate reductase (DHFR, U.S. Pat. Nos.4,399,216; 4,634,665; 4,656,134; 4,956,288; 5,149,636; 5,179,017),ampicillin, neomycin (G418), mycophenolic acid, or glutamine synthetase(GS, U.S. Pat. Nos. 5,122,464; 5,770,359; 5,827,739) resistance foreukaryotic cell culture, and tetracycline or ampicillin resistance genesfor culturing in E. coli and other bacteria or prokaryotics (the abovepatents are entirely incorporated hereby by reference). Appropriateculture mediums and conditions for the above-described host cells areknown in the art. Suitable vectors will be readily apparent to theskilled artisan. Introduction of a vector construct into a host cell canbe effected by calcium phosphate transfection, DEAE-dextran mediatedtransfection, cationic lipid-mediated transfection, electroporation,transduction, infection or other known methods. Such methods aredescribed in the art, such as Sambrook, supra, Chapters 1-4 and 16-18;Ausubel, supra, Chapters 1, 9, 13, 15, 16.

[0088] At least one antibody of the present invention can be expressedin a modified form, such as a fusion protein, and can include not onlysecretion signals, but also additional heterologous functional regions.For instance, a region of additional amino acids, particularly chargedamino acids, can be added to the N-terminus of an antibody to improvestability and persistence in the host cell, during purification, orduring subsequent handling and storage. Also, peptide moieties can beadded to an antibody of the present invention to facilitatepurification. Such regions can be removed prior to final preparation ofan antibody or at least one fragment thereof. Such methods are describedin many standard laboratory manuals, such as Sambrook, supra, Chapters17.29-17.42 and 18.1-18.74; Ausubel, supra, Chapters 16, 17 and 18.

[0089] Those of ordinary skill in the art are knowledgeable in thenumerous expression systems available for expression of a nucleic acidencoding a protein of the present invention.

[0090] Alternatively, nucleic acids of the present invention can beexpressed in a host cell by turning on (by manipulation) in a host cellthat contains endogenous DNA encoding an antibody of the presentinvention. Such methods are well known in the art, e.g., as described inU.S. Pat. Nos. 5,580,734, 5,641,670, 5,733,746, and 5,733,761, entirelyincorporated herein by reference.

[0091] Illustrative of cell cultures useful for the production of theantibodies, specified portions or variants thereof, are mammalian cells.Mammalian cell systems often will be in the form of monolayers of cellsalthough mammalian cell suspensions or bioreactors can also be used. Anumber of suitable host cell lines capable of expressing intactglycosylated proteins have been developed in the art, and include theCOS-1 (e.g., ATCC CRL 1650), COS-7 (e.g., ATCC CRL-1651), HEK293, BHK21(e.g., ATCC CRL-10), CHO (e.g., ATCC CRL 1610) and BSC-1 (e.g., ATCCCRL-26) cell lines, Cos-7 cells, CHO cells, hep G2 cells, P3X63Ag8.653,SP2/0-Ag14, 293 cells, HeLa cells and the like, which are readilyavailable from, for example, American Type Culture Collection, Manassas,Va. (www.atcc.org). Preferred host cells include cells of lymphoidorigin such as myeloma and lymphoma cells. Particularly preferred hostcells are P3X63Ag8.653 cells (ATCC Accession Number CRL-1580) andSP2/0-Ag14 cells (ATCC Accession Number CRL-1851). In a particularlypreferred embodiment, the recombinant cell is a P3X63Ab8.653 or aSP2/0-Ag14 cell.

[0092] Expression vectors for these cells can include one or more of thefollowing expression control sequences, such as, but not limited to anorigin of replication; a promoter (e.g., late or early SV40 promoters,the CMV promoter (U.S. Pat. Nos. 5,168,062; 5,385,839), an HSV tkpromoter, a pgk (phosphoglycerate kinase) promoter, an EF-1 alphapromoter (U.S. Pat. No. 5,266,491), at least one human immunoglobulinpromoter; an enhancer, and/or processing information sites, such asribosome binding sites, RNA splice sites, polyadenylation sites (e.g.,an SV40 large T Ag poly A addition site), and transcriptional terminatorsequences. See, e.g., Ausubel et al., supra; Sambrook, et al., supra.Other cells useful for production of nucleic acids or proteins of thepresent invention are known and/or available, for instance, from theAmerican Type Culture Collection Catalogue of Cell Lines and Hybridomas(www.atcc.org) or other known or commercial sources.

[0093] When eukaryotic host cells are employed, polyadenlyation ortranscription terminator sequences are typically incorporated into thevector. An example of a terminator sequence is the polyadenlyationsequence from the bovine growth hormone gene. Sequences for accuratesplicing of the transcript can also be included. An example of asplicing sequence is the VP 1 intron from SV40 (Sprague, et al., J.Virol. 45:773-781 (1983)). Additionally, gene sequences to controlreplication in the host cell can be incorporated into the vector, asknown in the art.

[0094] Purification of an Antibody

[0095] An anti-IL-12 antibody can be recovered and purified fromrecombinant cell cultures by well-known methods including, but notlimited to, protein A purification, ammonium sulfate or ethanolprecipitation, acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography and lectinchromatography. High performance liquid chromatography (“HPLC”) can alsobe employed for purification. See, e.g., Colligan, Current Protocols inImmunology, or Current Protocols in Protein Science, John Wiley & Sons,NY, N.Y., (1997-2001), e.g., Chapters 1, 4, 6, 8, 9, 10, each entirelyincorporated herein by reference.

[0096] Antibodies of the present invention include naturally purifiedproducts, products of chemical synthetic procedures, and productsproduced by recombinant techniques from a eukaryotic host, including,for example, yeast, higher plant, insect and mammalian cells. Dependingupon the host employed in a recombinant production procedure, theantibody of the present invention can be glycosylated or can benon-glycosylated, with glycosylated preferred. Such methods aredescribed in many standard laboratory manuals, such as Sambrook, supra,Sections 17.37-17.42; Ausubel, supra, Chapters 10, 12, 13, 16, 18 and20, Colligan, Protein Science, supra, Chapters 12-14, all entirelyincorporated herein by reference.

[0097] Anti-1L-12 Antibodies

[0098] The isolated antibodies of the present invention comprise anantibody encoded by any one of the polynucleotides of the presentinvention as discussed more fully herein, or any isolated or preparedantibody. Preferably, the human antibody or antigen-binding fragmentbinds human IL-12 and, thereby partially or substantially neutralizes atleast one biological activity of the protein. An antibody, or specifiedportion or variant thereof, that partially or preferably substantiallyneutralizes at least one biological activity of at least one IL-12protein or fragment can bind the protein or fragment and thereby inhibitactivitys mediated through the binding of IL-12 to the IL-12 receptor orthrough other IL-12-dependent or mediated mechanisms. As used herein,the term “neutralizing antibody” refers to an antibody that can inhibitan IL-12-dependent activity by about 20-120%, preferably by at leastabout 10, 20, 30, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93,94, 95, 96, 97, 98, 99, 100% or more depending on the assay. Thecapacity of an anti-IL-12 antibody to inhibit an IL-12-dependentactivity is preferably assessed by at least one suitable IL-12 proteinor receptor assay, as described herein and/or as known in the art. Ahuman antibody of the invention can be of any class (IgG, IgA, IgM, IgE,IgD, etc.) or isotype and can comprise a kappa or lambda light chain. Inone embodiment, the human antibody comprises an IgG heavy chain ordefined fragment, for example, at least one of isotypes, IgG1, IgG2,IgG3 or IgG4. Antibodies of this type can be prepared by employing atransgenic mouse or other trangenic non-human mammal comprising at leastone human light chain (e.g., IgG, IgA and IgM (e.g., γ1, γ2, γ3, γ4)transgenes as described herein and/or as known in the art. In anotherembodiment, the anti-human IL-12 human antibody comprises an IgG1 heavychain and a IgG1 light chain.

[0099] At least one antibody of the invention binds at least onespecified epitope specific to at least one IL-12 protein, subunit,fragment, portion or any combination thereof. The at least one epitopecan comprise at least one antibody binding region that comprises atleast one portion of said protein, which epitope is preferably comprisedof at least one extracellular, soluble, hydrophillic, external orcytoplasmic portion of said protein. The at least one specified epitopecan comprise any combination of at least one amino acid sequence of atleast 1-3 amino acids to the entire specified portion of contiguousamino acids of the SEQ ID NO:9.

[0100] Generally, the human antibody or antigen-binding fragment of thepresent invention will comprise an antigen-binding region that comprisesat least one human complementarity determining region (CDR1, CDR2 andCDR3) or variant of at least one heavy chain variable region and atleast one human complementarity determining region (CDR1, CDR2 and CDR3)or variant of at least one light chain variable region. As anon-limiting example, the antibody or antigen-binding portion or variantcan comprise at least one of the heavy chain CDR3 having the amino acidsequence of SEQ ID NO:3, and/or a light chain CDR3 having the amino acidsequence of SEQ ID NO:6. In a particular embodiment, the antibody orantigen-binding fragment can have an antigen-binding region thatcomprises at least a portion of at least one heavy chain CDR (i.e.,CDR1, CDR2 and/or CDR3) having the amino acid sequence of thecorresponding CDRs 1, 2 and/or 3 (e.g., SEQ ID NOS:1, 2, and/or 3). Inanother particular embodiment, the antibody or antigen-binding portionor variant can have an antigen-binding region that comprises at least aportion of at least one light chain CDR (i.e., CDR1, CDR2 and/or CDR3)having the amino acid sequence of the corresponding CDRs 1, 2 and/or 3(e.g., SEQ ID NOS: 4, 5, and/or 6). In a preferred embodiment the threeheavy chain CDRs and the three light chain CDRs of the anitbody orantigen-binding fragment have the amino acid sequence of thecorresponding CDR of at least one of mAb 12B75, C340, or any others asdescribed herein. Such antibodies can be prepared by chemically joiningtogether the various portions (e.g., CDRs, framework) of the antibodyusing conventional techniques, by preparing and expressing a (i.e., oneor more) nucleic acid molecule that encodes the antibody usingconventional techniques of recombinant DNA technology or by using anyother suitable method.

[0101] The anti-IL-12 antibody can comprise at least one of a heavy orlight chain variable region having a defined amino acid sequence. Forexample, in a preferred embodiment, the anti-IL-12 antibody comprises atleast one of at least one heavy chain variable region, optionally havingthe amino acid sequence of SEQ ID NO:7 and/or at least one light chainvariable region, optionally having the amino acid sequence of SEQ IDNO:8. antibodies that bind to human IL-12 and that comprise a definedheavy or light chain variable region can be prepared using suitablemethods, such as phage display (Katsube, Y., et al., Int J Mol. Med,1(5):863-868 (1998)) or methods that employ transgenic animals, as knownin the art and/or as described herein. For example, a transgenic mouse,comprising a functionally rearranged human immunoglobulin heavy chaintransgene and a transgene comprising DNA from a human immunoglobulinlight chain locus that can undergo functional rearrangement, can beimmunized with human IL-12 or a fragment thereof to elicit theproduction of antibodies. If desired, the antibody producing cells canbe isolated and hybridomas or other immortalized antibody-producingcells can be prepared as described herein and/or as known in the art.Alternatively, the antibody, specified portion or variant can beexpressed using the encoding nucleic acid or portion thereof in asuitable host cell.

[0102] The invention also relates to antibodies, antigen-bindingfragments, immunoglobulin chains and CDRs comprising amino acids in asequence that is substantially the same as an amino acid sequencedescribed herein. Preferably, such antibodies or antigen-bindingfragments and antibodies comprising such chains or CDRs can bind humanIL-12 with high affinity (e.g., K_(D) less than or equal to about 10⁻⁹M). Amino acid sequences that are substantially the same as thesequences described herein include sequences comprising conservativeamino acid substitutions, as well as amino acid deletions and/orinsertions. A conservative amino acid substitution refers to thereplacement of a first amino acid by a second amino acid that haschemical and/or physical properties (e.g, charge, structure, polarity,hydrophobicity/hydrophilicity) that are similar to those of the firstamino acid. Conservative substitutions include replacement of one aminoacid by another within the following groups: lysine (K), arginine (R)and histidine (H); aspartate (D) and glutamate (E); asparagine (N),glutamine (Q), serine (S), threonine (T), tyrosine (Y), K, R, H, D andE; alanine (A), valine (V), leucine (L), isoleucine (I), proline (P),phenylalanine (F), tryptophan (W), methionine (M), cysteine (C) andglycine (G); F, W and Y; C, S and T.

[0103] Amino Acid Codes

[0104] The amino acids that make up anti-IL-12 antibodies of the presentinvention are often abbreviated. The amino acid designations can beindicated by designating the amino acid by its single letter code, itsthree letter code, name, or three nucleotide codon(s) as is wellunderstood in the art (see Alberts, B., et al., Molecular Biology of TheCell, Third Ed., Garland Publishing, Inc., New York, 1994): SINGLE THREELETTER LETTER THREE NUCLEOTIDE CODE CODE NAME CODON(S) A Ala AlanineGCA, GCC, GCG, GCU C Cys Cysteine UGC, UGU D Asp Aspartic acid GAC, GAUE Glu Glutamic acid GAA, GAG F Phe Phenylanine UUC, UUU G Gly GlycineGGA, GGC, GGG, GGU H His Histidine CAC, CAU I Ile Isoleucine AUA, AUC,AUU K Lys Lysine AAA, AAG L Leu Leucine UUA, UUG, CUA, CUC, CUG, CUU MMet Methionine AUG N Asn Asparagine AAC, AAU P Pro Proline CCA, CCC,CCG, CCU Q Gln Glutamine CAA, CAG R Arg Arginine AGA, AGG, CGA, CGC,CGG, CGU S Ser Serine AGC, AGU, UCA, UCC, UCG, UCU T Thr Threonine ACA,ACC, ACG, ACU V Val Valine GUA, GUC, GUG, GUU W Trp Tryptophan UGG Y TyrTyrosine UAC, UAU

[0105] An anti-IL-12 antibody of the present invention can include oneor more amino acid substitutions, deletions or additions, either fromnatural mutations or human manipulation, as specified herein.

[0106] Of course, the number of amino acid substitutions a skilledartisan would make depends on many factors, including those describedabove. Generally speaking, the number of amino acid substitutions,insertions or deletions for any given anti-IL-12 Ig-derived protein,fragment or variant will not be more than 40, 30, 20, 19, 18, 17, 16,15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, such as 1-30 or anyrange or value therein, as specified herein.

[0107] Amino acids in an anti-IL-12 antibody of the present inventionthat are essential for function can be identified by methods known inthe art, such as site-directed mutagenesis or alanine-scanningmutagenesis (e.g., Ausubel, supra, Chapters 8, 15; Cunningham and Wells,Science 244:1081-1085 (1989)). The latter procedure introduces singlealanine mutations at every residue in the molecule. The resulting mutantmolecules are then tested for biological activity, such as, but notlimited to at least one IL-12 neutralizing activity. Sites that arecritical for antibody binding can also be identified by structuralanalysis such as crystallization, nuclear magnetic resonance orphotoaffinity labeling (Smith, et al., J. Mol. Biol. 224:899-904 (1992)and de Vos, et al., Science 255:306-312 (1992)).

[0108] Anti-IL-12 antibodies of the present invention can include, butare not limited to, at least one portion, sequence or combinationselected from 5 to all of the contiguous amino acids of at least one ofSEQ ID NOS:1, 2, 3, 4, 5, 6.

[0109] IL-12 antibodies or specified portions or variants of the presentinvention can include, but are not limited to, at least one portion,sequence or combination selected from at least 3-5 contiguous aminoacids of SEQ ID NO:1, 5-17 contiguous amino acids of SEQ ID NO:2, 5-10contiguous amino acids of SEQ ID NO:3, 5-11 contiguous amino acids ofSEQ ID NO:4, 5-7 contiguous amino acids of SEQ ID NO:5; 5-9 contiguousamino acids of SEQ ID NO:6; Leu21, Lys76, Met83, Ser85 of SEQ ID NO:7.

[0110] A(n) anti-IL-12 antibody can further optionally comprise apolypeptide of at least one of 70-100% of 5, 17, 10, 11, 7, 9, 119, or108 contiguous amino acids of at least one of SEQ ID NOS:1, 2, 3, 4, 5,6, 7 or 8.

[0111] In one embodiment, the amino acid sequence of an immunoglobulinchain, or portion thereof (e.g., variable region, CDR) has about 70-100%identity (e.g., 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 orany range or value therein) to the amino acid sequence of thecorresponding chain of at least one of SEQ ID NOS:7, 8. For example, theamino acid sequence of a light chain variable region can be comparedwith the sequence of SEQ ID NO:8, or the amino acid sequence of a heavychain CDR3 can be compared with SEQ ID NO:3. Preferably, 70-100% aminoacid identity (i.e., 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or anyrange or value therein) is determined using a suitable computeralgorithm, as known in the art.

[0112] Exemplary heavy chain and light chain variable regions sequencesare provided in SEQ ID NOS: 7 and 8. The antibodies of the presentinvention, or specified variants thereof, can comprise any number ofcontiguous amino acid residues from an antibody of the presentinvention, wherein that number is selected from the group of integersconsisting of from 10-100% of the number of contiguous residues in ananti-IL-12 antibody. Optionally, this subsequence of contiguous aminoacids is at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110,120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250 ormore amino acids in length, or any range or value therein. Further, thenumber of such subsequences can be any integer selected from the groupconsisting of from 1 to 20, such as at least 2, 3, 4, or 5.

[0113] As those of skill will appreciate, the present invention includesat least one biologically active antibody of the present invention.Biologically active antibodies have a specific activity at least 20%,30%, or 40%, and preferably at least 50%, 60%, or 70%, and mostpreferably at least 80%, 90%, or 95%-1000% of that of the native(non-synthetic), endogenous or related and known antibody. Methods ofassaying and quantifying measures of enzymatic activity and substratespecificity, are well known to those of skill in the art.

[0114] In another aspect, the invention relates to human antibodies andantigen-binding fragments, as described herein, which are modified bythe covalent attachment of an organic moiety. Such modification canproduce an antibody or antigen-binding fragment with improvedpharmacokinetic properties (e.g., increased in vivo serum half-life).The organic moiety can be a linear or branched hydrophilic polymericgroup, fatty acid group, or fatty acid ester group. In particularembodiments, the hydrophilic polymeric group can have a molecular weightof about 800 to about 120,000 Daltons and can be a polyalkane glycol(e.g., polyethylene glycol (PEG), polypropylene glycol (PPG)),carbohydrate polymer, amino acid polymer or polyvinyl pyrolidone, andthe fatty acid or fatty acid ester group can comprise from about eightto about forty carbon atoms.

[0115] The modified antibodies and antigen-binding fragments of theinvention can comprise one or more organic moieties that are covalentlybonded, directly or indirectly, to the antibody. Each organic moietythat is bonded to an antibody or antigen-binding fragment of theinvention can independently be a hydrophilic polymeric group, a fattyacid group or a fatty acid ester group. As used herein, the term “fattyacid” encompasses mono-carboxylic acids and di-carboxylic acids. A“hydrophilic polymeric group,” as the term is used herein, refers to anorganic polymer that is more soluble in water than in octane. Forexample, polylysine is more soluble in water than in octane. Thus, anantibody modified by the covalent attachment of polylysine isencompassed by the invention. Hydrophilic polymers suitable formodifying antibodies of the invention can be linear or branched andinclude, for example, polyalkane glycols (e.g., PEG,monomethoxy-polyethylene glycol (mPEG), PPG and the like), carbohydrates(e.g., dextran, cellulose, oligosaccharides, polysaccharides and thelike), polymers of hydrophilic amino acids (e.g., polylysine,polyarginine, polyaspartate and the like), polyalkane oxides (e.g.,polyethylene oxide, polypropylene oxide and the like) and polyvinylpyrolidone. Preferably, the hydrophilic polymer that modifies theantibody of the invention has a molecular weight of about 800 to about150,000 Daltons as a separate molecular entity. For example PEG₅₀₀₀ andPEG_(20,000) wherein the subscript is the average molecular weight ofthe polymer in Daltons, can be used. The hydrophilic polymeric group canbe substituted with one to about six alkyl, fatty acid or fatty acidester groups. Hydrophilic polymers that are substituted with a fattyacid or fatty acid ester group can be prepared by employing suitablemethods. For example, a polymer comprising an amine group can be coupledto a carboxylate of the fatty acid or fatty acid ester, and an activatedcarboxylate (e.g., activated with N,N-carbonyl diimidazole) on a fattyacid or fatty acid ester can be coupled to a hydroxyl group on apolymer.

[0116] Fatty acids and fatty acid esters suitable for modifyingantibodies of the invention can be saturated or can contain one or moreunits of unsaturation. Fatty acids that are suitable for modifyingantibodies of the invention include, for example, n-dodecanoate (C₁₂,laurate), n-tetradecanoate (C₁₄, myristate), n-octadecanoate (C₁₈,stearate), n-eicosanoate (C₂₀, arachidate), n-docosanoate (C₂₂,behenate), n-triacontanoate (C₃₀), n-tetracontanoate (C₄₀),cis-Δ9-octadecanoate (C₁₈, oleate), all cis-Δ5,8,11,14-eicosatetraenoate(C₂₀, arachidonate), octanedioic acid, tetradecanedioic acid,octadecanedioic acid, docosanedioic acid, and the like. Suitable fattyacid esters include mono-esters of dicarboxylic acids that comprise alinear or branched lower alkyl group. The lower alkyl group can comprisefrom one to about twelve, preferably one to about six, carbon atoms.

[0117] The modified human antibodies and antigen-binding fragments canbe prepared using suitable methods, such as by reaction with one or moremodifying agents. A “modifying agent” as the term is used herein, refersto a suitable organic group (e.g., hydrophilic polymer, a fatty acid, afatty acid ester) that comprises an activating group. An “activatinggroup” is a chemical moiety or functional group that can, underappropriate conditions, react with a second chemical group therebyforming a covalent bond between the modifying agent and the secondchemical group. For example, amine-reactive activating groups includeelectrophilic groups such as tosylate, mesylate, halo (chloro, bromo,fluoro, iodo), N-hydroxysuccinimidyl esters (NHS), and the like.Activating groups that can react with thiols include, for example,maleimide, iodoacetyl, acrylolyl, pyridyl disulfides,5-thiol-2-nitrobenzoic acid thiol (TNB-thiol), and the like. An aldehydefunctional group can be coupled to amine- or hydrazide-containingmolecules, and an azide group can react with a trivalent phosphorousgroup to form phosphoramidate or phosphorimide linkages. Suitablemethods to introduce activating groups into molecules are known in theart (see for example, Hermanson, G. T., Bioconjugate Techniques,Academic Press: San Diego, Calif. (1996)). An activating group can bebonded directly to the organic group (e.g., hydrophilic polymer, fattyacid, fatty acid ester), or through a linker moiety, for example adivalent C₁-C₁₂ group wherein one or more carbon atoms can be replacedby a heteroatom such as oxygen, nitrogen or sulfur. Suitable linkermoieties include, for example, tetraethylene glycol, —(CH₂)₃—,—NH—(CH₂)₆—NH—, —(CH₂)₂—NH— and —CH₂—O—CH₂—CH₂—O—CH₂—CH₂—O—CH—NH—.Modifying agents that comprise a linker moiety can be produced, forexample, by reacting a mono-Boc-alkyldiamine (e.g.,mono-Boc-ethylenediamine, mono-Boc-diaminohexane) with a fatty acid inthe presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) toform an amide bond between the free amine and the fatty acidcarboxylate. The Boc protecting group can be removed from the product bytreatment with trifluoroacetic acid (TFA) to expose a primary amine thatcan be coupled to another carboxylate as described, or can be reactedwith maleic anhydride and the resulting product cyclized to produce anactivated maleimido derivative of the fatty acid. (See, for example,Thompson, et al., WO 92/16221 the entire teachings of which areincorporated herein by reference.)

[0118] The modified antibodies of the invention can be produced byreacting a human antibody or antigen-binding fragment with a modifyingagent. For example, the organic moieties can be bonded to the antibodyin a non-site specific manner by employing an amine-reactive modifyingagent, for example, an NHS ester of PEG. Modified human antibodies orantigen-binding fragments can also be prepared by reducing disulfidebonds (e.g., intra-chain disulfide bonds) of an antibody orantigen-binding fragment. The reduced antibody or antigen-bindingfragment can then be reacted with a thiol-reactive modifying agent toproduce the modified antibody of the invention. Modified humanantibodies and antigen-binding fragments comprising an organic moietythat is bonded to specific sites of an antibody of the present inventioncan be prepared using suitable methods, such as reverse proteolysis(Fisch et al., Bioconjugate Chem., 3:147-153 (1992); Werlen et al.,Bioconjugate Chem., 5:411-417 (1994); Kumaran et al., Protein Sci.6(10):2233-2241 (1997); Itoh et al., Bioorg. Chem., 24(1): 59-68 (1996);Capellas et al., Biotechnol. Bioeng., 56(4):456-463 (1997)), and themethods described in Hermanson, G. T., Bioconjugate Techniques, AcademicPress: San Diego, Calif. (1996).

[0119] Anti-Idiotype Antibodies to Anti-IL-12 IG Derived ProteinCompositions

[0120] In addition to monoclonal or chimeric anti-IL-12 antibodies, thepresent invention is also directed to an anti-idiotypic (anti-Id)antibody specific for such antibodies of the invention. An anti-Idantibody is an antibody which recognizes unique determinants generallyassociated with the antigen-binding region of another antibody. Theanti-Id can be prepared by immunizing an animal of the same species andgenetic type (e.g. mouse strain) as the source of the Id antibody withthe antibody or a CDR containing region thereof. The immunized animalwill recognize and respond to the idiotypic determinants of theimmunizing antibody and produce an anti-Id antibody. The anti-Idantibody may also be used as an “immunogen” to induce an immune responsein yet another animal, producing a so-called anti-anti-Id antibody.

[0121] Anti-IL-12 IG Derived Protein Compositions

[0122] The present invention also provides at least one anti-IL-12antibody composition comprising at least one, at least two, at leastthree, at least four, at least five, at least six or more anti-IL-12antibodies thereof, as described herein and/or as known in the art thatare provided in a non-naturally occurring composition, mixture or form.Such compositions comprise non-naturally occurring compositionscomprising at least one or two full length, C- and/or N-terminallydeleted variants, domains, fragments, or specified variants, of theanti-IL-12 antibody amino acid sequence selected from the groupconsisting of 70-100% of the contiguous amino acids of SEQ ID NOS:1, 2,3, 4, 5, 6, 7 or 8, or specified fragments, domains or variants thereof.Preferred anti-IL-12 derived protein, fragment or variant compositionsinclude at least one or two full length, fragments, domains or variantsas at least one CDR containing portions of the anti-IL-12 antibodysequence of 70-100% of SEQ ID NOS: 1, 2, 3, 4, 5, 6, or specifiedfragments, domains or variants thereof. Further preferred compositionscomprise 40-99% of at least one of 70-100% of SEQ ID NOS: 1, 2, 3, 4, 5,6, or specified fragments, domains or variants thereof. Such compositionpercentages are by weight, volume, concentration, molarity, or molalityas liquid or dry solutions, mixtures, suspension, emulsions or colloids,as known in the art or as described herein.

[0123] Anti-IL-12 antibody compositions of the present invention canfurther comprise at least one of any suitable and effective amount of acomposition or pharmaceutical composition comprising at least oneanti-IL-12 antibody to a cell, tissue, organ, animal or patient in needof such modulation, treatment or therapy, optionally further comprisingat least one selected from at least one TNF antagonist (e.g., but notlimited to a TNF antibody or fragment, a soluble TNF receptor orfragment, fusion proteins thereof, or a small molecule TNF antagonist),an antirheumatic (e.g., methotrexate, auranofin, aurothioglucose,azathioprine, etanercept, gold sodium thiomalate, hydroxychloroquinesulfate, leflunomide, sulfasalzine), a muscle relaxant, a narcotic, anon-steroid anti-inflammatory drug (NSAID), an analgesic, an anesthetic,a sedative, a local anethetic, a neuromuscular blocker, an antimicrobial(e.g., aminoglycoside, an antifungal, an antiparasitic, an antiviral, acarbapenem, cephalosporin, a flurorquinolone, a macrolide, a penicillin,a sulfonamide, a tetracycline, another antimicrobial), an antipsoriatic,a corticosteriod, an anabolic steroid, a diabetes related agent, amineral, a nutritional, a thyroid agent, a vitamin, a calcium relatedhormone, an antidiarrheal, an antitussive, an antiemetic, an antiulcer,a laxative, an anticoagulant, an erythropieitin (e.g., epoetin alpha), afilgrastim (e.g., G-CSF, Neupogen), a sargramostim (GM-CSF, Leukine), animmunization, an immunoglobulin, an immunosuppressive (e.g.,basiliximab, cyclosporine, daclizumab), a growth hormone, a hormonereplacement drug, an estrogen receptor modulator, a mydriatic, acycloplegic, an alkylating agent, an antimetabolite, a mitoticinhibitor, a radiopharmaceutical, an antidepressant, antimanic agent, anantipsychotic, an anxiolytic, a hypnotic, a sympathomimetic, astimulant, donepezil, tacrine, an asthma medication, a beta agonist, aninhaled steroid, a leukotriene inhibitor, a methylxanthine, a cromolyn,an epinephrine or analog, dornase alpha (Pulmozyme), a cytokine or acytokine antagonist. Non-limiting examples of such cytokines include,but are not limited to, any of IL-1 to IL-23. Suitable dosages are wellknown in the art. See, e.g., Wells et al., eds., PharmacotherapyHandbook, 2^(nd) Edition, Appleton and Lange, Stamford, Conn. (2000);PDR Pharmacopoeia, Tarascon Pocket Pharmacopoeia 2000, Deluxe Edition,Tarascon Publishing, Loma Linda, Calif. (2000), each of which referencesare entirely incorporated herein by reference.

[0124] Such anti-cancer or anti-infectives can also include toxinmolecules that are associated, bound, co-formulated or co-administeredwith at least one antibody of the present invention. The toxin canoptionally act to selectively kill the pathologic cell or tissue. Thepathologic cell can be a cancer or other cell. Such toxins can be, butare not limited to, purified or recombinant toxin or toxin fragmentcomprising at least one functional cytotoxic domain of toxin, e.g.,selected from at least one of ricin, diphtheria toxin, a venom toxin, ora bacterial toxin. The term toxin also includes both endotoxins andexotoxins produced by any naturally occurring, mutant or recombinantbacteria or viruses which may cause any pathological condition in humansand other mammals, including toxin shock, which can result in death.Such toxins may include, but are not limited to, enterotoxigenic E. coliheat-labile enterotoxin (LT), heat-stable enterotoxin (ST), Shigellacytotoxin, Aeromonas enterotoxins, toxic shock syndrome toxin-1(TSST-1), Staphylococcal enterotoxin A (SEA), B (SEB), or C (SEC),Streptococcal enterotoxins and the like. Such bacteria include, but arenot limited to, strains of a species of enterotoxigenic E. coli (ETEC),enterohemorrhagic E. coli (e.g., strains of serotype 0157:H7),Staphylococcus species (e.g., Staphylococcus aureus, Staphylococcuspyogenes), Shigella species (e.g., Shigella dysenteriae, Shigellaflexneri, Shigella boydii, and Shigella sonnei), Salmonella species(e.g., Salmonella typhi, Salmonella cholera-suis, Salmonellaenteritidis), Clostridium species (e.g., Clostridium perfringens,Clostridium dificile, Clostridium botulinum), Camphlobacter species(e.g., Camphlobacter jejuni, Camphlobacter fetus), Heliobacter species,(e.g., Heliobacter pylori), Aeromonas species (e.g., Aeromonas sobria,Aeromonas hydrophila, Aeromonas caviae), Pleisomonas shigelloides,Yersina enterocolitica, Vibrios species (e.g., Vibrios cholerae, Vibriosparahemolyticus), Klebsiella species, Pseudomonas aeruginosa, andStreptococci. See, e.g., Stein, ed., INTERNAL MEDICINE, 3rd ed., pp1-13, Little, Brown and Co., Boston, (1990); Evans et al., eds.,Bacterial Infections of Humans: Epidemiology and Control, 2d. Ed., pp239-254, Plenum Medical Book Co., New York (1991); Mandell et al,Principles and Practice of Infectious Diseases, 3d. Ed., ChurchillLivingstone, New York (1990); Berkow et al, eds., The Merck Manual, 16thedition, Merck and Co., Rahway, N.J., 1992; Wood et al, FEMSMicrobiology Immunology, 76:121-134 (1991); Marrack et al, Science,248:705-711 (1990), the contents of which references are incorporatedentirely herein by reference.

[0125] Anti-IL-12 antibody compounds, compositions or combinations ofthe present invention can further comprise at least one of any suitableauxiliary, such as, but not limited to, diluent, binder, stabilizer,buffers, salts, lipophilic solvents, preservative, adjuvant or the like.Pharmaceutically acceptable auxiliaries are preferred. Non-limitingexamples of, and methods of preparing such sterile solutions are wellknown in the art, such as, but limited to, Gennaro, Ed., Remington 'sPharmaceutical Sciences, 18^(th) Edition, Mack Publishing Co. (Easton,Pa.) 1990. Pharmaceutically acceptable carriers can be routinelyselected that are suitable for the mode of administration, solubilityand/or stability of the anti-IL-12 antibody, fragment or variantcomposition as well known in the art or as described herein.

[0126] Pharmaceutical excipients and additives useful in the presentcomposition include but are not limited to proteins, peptides, aminoacids, lipids, and carbohydrates (e.g., sugars, includingmonosaccharides, di-, tri-, tetra-, and oligosaccharides; derivatizedsugars such as alditols, aldonic acids, esterified sugars and the like;and polysaccharides or sugar polymers), which can be present singly orin combination, comprising alone or in combination 1-99.99% by weight orvolume. Exemplary protein excipients include serum albumin such as humanserum albumin (HSA), recombinant human albumin (rHA), gelatin, casein,and the like. Representative amino acid/antibody components, which canalso function in a buffering capacity, include alanine, glycine,arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine,lysine, leucine, isoleucine, valine, methionine, phenylalanine,aspartame, and the like. One preferred amino acid is glycine.

[0127] Carbohydrate excipients suitable for use in the inventioninclude, for example, monosaccharides such as fructose, maltose,galactose, glucose, D-mannose, sorbose, and the like; disaccharides,such as lactose, sucrose, trehalose, cellobiose, and the like;polysaccharides, such as raffinose, melezitose, maltodextrins, dextrans,starches, and the like; and alditols, such as mannitol, xylitol,maltitol, lactitol, xylitol sorbitol (glucitol), myoinositol and thelike. Preferred carbohydrate excipients for use in the present inventionare mannitol, trehalose, and raffinose.

[0128] Anti-IL-12 antibody compositions can also include a buffer or apH adjusting agent; typically, the buffer is a salt prepared from anorganic acid or base. Representative buffers include organic acid saltssuch as salts of citric acid, ascorbic acid, gluconic acid, carbonicacid, tartaric acid, succinic acid, acetic acid, or phthalic acid; Tris,tromethamine hydrochloride, or phosphate buffers. Preferred buffers foruse in the present compositions are organic acid salts such as citrate.

[0129] Additionally, anti-IL-12 antibody compositions of the inventioncan include polymeric excipients/additives such aspolyvinylpyrrolidones, ficolls (a polymeric sugar), dextrates (e.g.,cyclodextrins, such as 2-hydroxypropyl-β-cyclodextrin), polyethyleneglycols, flavoring agents, antimicrobial agents, sweeteners,antioxidants, antistatic agents, surfactants (e.g., polysorbates such as“TWEEN 20” and “TWEEN 80”), lipids (e.g., phospholipids, fatty acids),steroids (e.g., cholesterol), and chelating agents (e.g., EDTA).

[0130] These and additional known pharmaceutical excipients and/oradditives suitable for use in the anti-IL-12 antibody, portion orvariant compositions according to the invention are known in the art,e.g., as listed in “Remington: The Science & Practice of Pharmacy”,19^(th) ed., Williams & Williams, (1995), and in the “Physician's DeskReference”, 52^(nd) ed., Medical Economics, Montvale, N.J. (1998), thedisclosures of which are entirely incorporated herein by reference.Preferrred carrier or excipient materials are carbohydrates (e.g.,saccharides and alditols) and buffers (e.g., citrate) or polymericagents.

[0131] Formulations

[0132] As noted above, the invention provides for stable formulations,which is preferably a phosphate buffer with saline or a chosen salt, aswell as preserved solutions and formulations containing a preservativeas well as multi-use preserved formulations suitable for pharmaceuticalor veterinary use, comprising at least one anti-IL-12 antibody in apharmaceutically acceptable formulation. Preserved formulations containat least one known preservative or optionally selected from the groupconsisting of at least one phenol, m-cresol, p-cresol, o-cresol,chlorocresol, benzyl alcohol, phenylmercuric nitrite, phenoxyethanol,formaldehyde, chlorobutanol, magnesium chloride (e.g., hexahydrate),alkylparaben (methyl, ethyl, propyl, butyl and the like), benzalkoniumchloride, benzethonium chloride, sodium dehydroacetate and thimerosal,or mixtures thereof in an aqueous diluent. Any suitable concentration ormixture can be used as known in the art, such as 0.001-5%, or any rangeor value therein, such as, but not limited to 0.001, 0.003, 0.005,0.009, 0.01, 0.02, 0.03, 0.05, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7,0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1,2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5,3.6, 3.7, 3.8, 3.9, 4.0, 4.3, 4.5, 4.6, 4.7, 4.8, 4.9, or any range orvalue therein. Non-limiting examples include, no preservative, 0.1-2%m-cresol (e.g., 0.2, 0.3, 0.4, 0.5, 0.9, 1.0%), 0.1-3% benzyl alcohol(e.g., 0.5, 0.9, 1.1., 1.5, 1.9, 2.0, 2.5%), 0.001-0.5% thimerosal(e.g., 0.005, 0.01), 0.001-2.0% phenol (e.g., 0.05, 0.25, 0.28, 0.5,0.9, 1.0%), 0.0005-1.0% alkylparaben(s) (e.g., 0.00075, 0.0009, 0.001,0.002, 0.005, 0.0075, 0.009, 0.01, 0.02, 0.05, 0.075, 0.09, 0.1, 0.2,0.3, 0.5, 0.75, 0.9, 1.0%), and the like.

[0133] As noted above, the invention provides an article of manufacture,comprising packaging material and at least one vial comprising asolution of at least one anti-IL-12 antibody with the prescribed buffersand/or preservatives, optionally in an aqueous diluent, wherein saidpackaging material comprises a label that indicates that such solutioncan be held over a period of 1, 2, 3, 4, 5, 6, 9, 12, 18, 20, 24, 30,36, 40, 48, 54, 60, 66, 72 hours or greater. The invention furthercomprises an article of manufacture, comprising packaging material, afirst vial comprising lyophilized at least one anti-IL-12 antibody, anda second vial comprising an aqueous diluent of prescribed buffer orpreservative, wherein said packaging material comprises a label thatinstructs a patient to reconstitute the at least one anti-IL-12 antibodyin the aqueous diluent to form a solution that can be held over a periodof twenty-four hours or greater.

[0134] The at least one anti-IL-12 antibody used in accordance with thepresent invention can be produced by recombinant means, including frommammalian cell or transgenic preparations, or can be purified from otherbiological sources, as described herein or as known in the art.

[0135] The range of at least one anti-IL-12 antibody in the product ofthe present invention includes amounts yielding upon reconstitution, ifin a wet/dry system, concentrations from about 1.0 μg/ml to about 1000mg/ml, although lower and higher concentrations are operable and aredependent on the intended delivery vehicle, e.g., solution formulationswill differ from transdermal patch, pulmonary, transmucosal, or osmoticor micro pump methods.

[0136] Preferably, the aqueous diluent optionally further comprises apharmaceutically acceptable preservative. Preferred preservativesinclude those selected from the group consisting of phenol, m-cresol,p-cresol, o-cresol, chlorocresol, benzyl alcohol, alkylparaben (methyl,ethyl, propyl, butyl and the like), benzalkonium chloride, benzethoniumchloride, sodium dehydroacetate and thimerosal, or mixtures thereof. Theconcentration of preservative used in the formulation is a concentrationsufficient to yield an anti-microbial effect. Such concentrations aredependent on the preservative selected and are readily determined by theskilled artisan.

[0137] Other excipients, e.g. isotonicity agents, buffers, antioxidants,preservative enhancers, can be optionally and preferably added to thediluent. An isotonicity agent, such as glycerin, is commonly used atknown concentrations. A physiologically tolerated buffer is preferablyadded to provide improved pH control. The formulations can cover a widerange of pHs, such as from about pH 4 to about pH 10, and preferredranges from about pH 5 to about pH 9, and a most preferred range ofabout 6.0 to about 8.0. Preferably the formulations of the presentinvention have pH between about 6.8 and about 7.8. Preferred buffersinclude phosphate buffers, most preferably sodium phosphate,particularly phosphate buffered saline (PBS).

[0138] Other additives, such as a pharmaceutically acceptablesolubilizers like Tween 20 (polyoxyethylene (20) sorbitan monolaurate),Tween 40 (polyoxyethylene (20) sorbitan monopalmitate), Tween 80(polyoxyethylene (20) sorbitan monooleate), Pluronic F68(polyoxyethylene polyoxypropylene block copolymers), and PEG(polyethylene glycol) or non-ionic surfactants such as polysorbate 20 or80 or poloxamer 184 or 188, Pluronic® polyls, other block co-polymers,and chelators such as EDTA and EGTA can optionally be added to theformulations or compositions to reduce aggregation. These additives areparticularly useful if a pump or plastic container is used to administerthe formulation. The presence of pharmaceutically acceptable surfactantmitigates the propensity for the protein to aggregate.

[0139] The formulations of the present invention can be prepared by aprocess which comprises mixing at least one anti-IL-12 antibody and apreservative selected from the group consisting of phenol, m-cresol,p-cresol, o-cresol, chlorocresol, benzyl alcohol, alkylparaben, (methyl,ethyl, propyl, butyl and the like), benzalkonium chloride, benzethoniumchloride, sodium dehydroacetate and thimerosal or mixtures thereof in anaqueous diluent. Mixing the at least one anti-IL-12 antibody andpreservative in an aqueous diluent is carried out using conventionaldissolution and mixing procedures. To prepare a suitable formulation,for example, a measured amount of at least one anti-IL-12 antibody inbuffered solution is combined with the desired preservative in abuffered solution in quantities sufficient to provide the protein andpreservative at the desired concentrations. Variations of this processwould be recognized by one of ordinary skill in the art. For example,the order the components are added, whether additional additives areused, the temperature and pH at which the formulation is prepared, areall factors that can be optimized for the concentration and means ofadministration used.

[0140] The claimed formulations can be provided to patients as clearsolutions or as dual vials comprising a vial of lyophilized at least oneanti-IL-12 antibody that is reconstituted with a second vial containingwater, a preservative and/or excipients, preferably a phosphate bufferand/or saline and a chosen salt, in an aqueous diluent. Either a singlesolution vial or dual vial requiring reconstitution can be reusedmultiple times and can suffice for a single or multiple cycles ofpatient treatment and thus can provide a more convenient treatmentregimen than currently available.

[0141] The present claimed articles of manufacture are useful foradministration over a period of immediately to twenty-four hours orgreater. Accordingly, the presently claimed articles of manufactureoffer significant advantages to the patient. Formulations of theinvention can optionally be safely stored at temperatures of from about2 to about 40° C. and retain the biologically activity of the proteinfor extended periods of time, thus, allowing a package label indicatingthat the solution can be held and/or used over a period of 6, 12, 18,24, 36, 48, 72, or 96 hours or greater. If preserved diluent is used,such label can include use up to 1-12 months, one-half, one and a half,and/or two years.

[0142] The solutions of at least one anti-IL-12 antibody in theinvention can be prepared by a process that comprises mixing at leastone antibody in an aqueous diluent. Mixing is carried out usingconventional dissolution and mixing procedures. To prepare a suitablediluent, for example, a measured amount of at least one antibody inwater or buffer is combined in quantities sufficient to provide theprotein and optionally a preservative or buffer at the desiredconcentrations. Variations of this process would be recognized by one ofordinary skill in the art. For example, the order the components areadded, whether additional additives are used, the temperature and pH atwhich the formulation is prepared, are all factors that can be optimizedfor the concentration and means of administration used.

[0143] The claimed products can be provided to patients as clearsolutions or as dual vials comprising a vial of lyophilized at least oneanti-IL-12 antibody that is reconstituted with a second vial containingthe aqueous diluent. Either a single solution vial or dual vialrequiring reconstitution can be reused multiple times and can sufficefor a single or multiple cycles of patient treatment and thus provides amore convenient treatment regimen than currently available.

[0144] The claimed products can be provided indirectly to patients byproviding to pharmacies, clinics, or other such institutions andfacilities, clear solutions or dual vials comprising a vial oflyophilized at least one anti-IL-12 antibody that is reconstituted witha second vial containing the aqueous diluent. The clear solution in thiscase can be up to one liter or even larger in size, providing a largereservoir from which smaller portions of the at least one antibodysolution can be retrieved one or multiple times for transfer intosmaller vials and provided by the pharmacy or clinic to their customersand/or patients.

[0145] Recognized devices comprising these single vial systems includethose pen-injector devices for delivery of a solution such as BD Pens,BD Autojector®, Humaject®, NovoPen®, B-D®Pen, AutoPen®, and OptiPen®,GenotropinPen®, Genotronorm Pen®, Humatro Pen®, Reco-Pen®, Roferon Pen®,Biojector®, iject®, J-tip Needle-Free Injector®, Intraject®, Medi-Ject®,e.g., as made or developed by Becton Dickensen (Franklin Lakes, N.J.,www.bectondickenson.com), Disetronic (Burgdorf, Switzerland,www.disetronic.com; Bioject, Portland, Oreg. (www.bioject.com); NationalMedical Products, Weston Medical (Peterborough, UK,www.weston-medical.com), Medi-Ject Corp (Minneapolis, Minn.,www.mediject.com). Recognized devices comprising a dual vial systeminclude those pen-injector systems for reconstituting a lyophilized drugin a cartridge for delivery of the reconstituted solution such as theHumatroPen®.

[0146] The products presently claimed include packaging material. Thepackaging material provides, in addition to the information required bythe regulatory agencies, the conditions under which the product can beused. The packaging material of the present invention providesinstructions to the patient to reconstitute the at least one anti-IL-12antibody in the aqueous diluent to form a solution and to use thesolution over a period of 2-24 hours or greater for the two vial,wet/dry, product. For the single vial, solution product, the labelindicates that such solution can be used over a period of 2-24 hours orgreater. The presently claimed products are useful for humanpharmaceutical product use.

[0147] The formulations of the present invention can be prepared by aprocess that comprises mixing at least one anti-IL-12 antibody and aselected buffer, preferably a phosphate buffer containing saline or achosen salt. Mixing the at least one antibody and buffer in an aqueousdiluent is carried out using conventional dissolution and mixingprocedures. To prepare a suitable formulation, for example, a measuredamount of at least one antibody in water or buffer is combined with thedesired buffering agent in water in quantities sufficient to provide theprotein and buffer at the desired concentrations. Variations of thisprocess would be recognized by one of ordinary skill in the art. Forexample, the order the components are added, whether additionaladditives are used, the temperature and pH at which the formulation isprepared, are all factors that can be optimized for the concentrationand means of administration used.

[0148] The claimed stable or preserved formulations can be provided topatients as clear solutions or as dual vials comprising a vial oflyophilized at least one anti-IL-12 antibody that is reconstituted witha second vial containing a preservative or buffer and excipients in anaqueous diluent. Either a single solution vial or dual vial requiringreconstitution can be reused multiple times and can suffice for a singleor multiple cycles of patient treatment and thus provides a moreconvenient treatment regimen than currently available.

[0149] At least one anti-IL-12 antibody in either the stable orpreserved formulations or solutions described herein, can beadministered to a patient in accordance with the present invention via avariety of delivery methods including SC or IM injection; transdermal,pulmonary, transmucosal, implant, osmotic pump, cartridge, micro pump,or other means appreciated by the skilled artisan, as well-known in theart.

[0150] Therapeutic Applications

[0151] The present invention also provides a method for modulating ortreating at least one immune related disease, in a cell, tissue, organ,animal, or patient including, but not limited to, at least one ofrheumatoid arthritis, juvenile rheumatoid arthritis, systemic onsetjuvenile rheumatoid arthritis, psoriatic arthritis, ankylosingspondilitis, gastric ulcer, seronegative arthropathies, osteoarthritis,inflammatory bowel disease, ulcerative colitis, systemic lupuserythematosis, antiphospholipid syndrome, iridocyclitis/uveitis/opticneuritis, idiopathic pulmonary fibrosis, systemic vasculitis/wegener'sgranulomatosis, sarcoidosis, orchitis/vasectomy reversal procedures,allergic/atopic diseases, asthma, allergic rhinitis, eczema, allergiccontact dermatitis, allergic conjunctivitis, hypersensitivitypneumonitis, transplants, organ transplant rejection, graft-versus-hostdisease, systemic inflammatory response syndrome, sepsis syndrome, grampositive sepsis, gram negative sepsis, culture negative sepsis, fungalsepsis, neutropenic fever, urosepsis, meningococcemia,trauma/hemorrhage, burns, ionizing radiation exposure, acutepancreatitis, adult respiratory distress syndrome, rheumatoid arthritis,alcohol-induced hepatitis, chronic inflammatory pathologies,sarcoidosis, Crohn's pathology, sickle cell anemia, diabetes, nephrosis,atopic diseases, hypersensitity reactions, allergic rhinitis, hay fever,perennial rhinitis, conjunctivitis, endometriosis, asthma, urticaria,systemic anaphalaxis, dermatitis, pernicious anemia, hemolyticdisesease, thrombocytopenia, graft rejection of any organ or tissue,kidney translplant rejection, heart transplant rejection, livertransplant rejection, pancreas transplant rejection, lung transplantrejection, bone marrow transplant (BMT) rejection, skin allograftrejection, cartilage transplant rejection, bone graft rejection, smallbowel transplant rejection, fetal thymus implant rejection, parathyroidtransplant rejection, xenograft rejection of any organ or tissue,allograft rejection, anti-receptor hypersensitivity reactions, Gravesdisease, Raynoud's disease, type B insulin-resistant diabetes, asthma,myasthenia gravis, antibody-meditated cytotoxicity, type IIIhypersensitivity reactions, systemic lupus erythematosus, POEMS syndrome(polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy,and skin changes syndrome), polyneuropathy, organomegaly,endocrinopathy, monoclonal gammopathy, skin changes syndrome,antiphospholipid syndrome, pemphigus, scleroderma, mixed connectivetissue disease, idiopathic Addison's disease, diabetes mellitus, chronicactive hepatitis, primary billiary cirrhosis, vitiligo, vasculitis,post-MI cardiotomy syndrome, type IV hypersensitivity, contactdermatitis, hypersensitivity pneumonitis, allograft rejection,granulomas due to intracellular organisms, drug sensitivity,metabolic/idiopathic, Wilson's disease, hemachromatosis,alpha-1-antitrypsin deficiency, diabetic retinopathy, hashimoto'sthyroiditis, osteoporosis, hypothalamic-pituitary-adrenal axisevaluation, primary biliary cirrhosis, thyroiditis, encephalomyelitis,cachexia, cystic fibrosis, neonatal chronic lung disease, chronicobstructive pulmonary disease (COPD), familial hematophagocyticlymphohistiocytosis, dermatologic conditions, psoriasis, alopecia,nephrotic syndrome, nephritis, glomerular nephritis, acute renalfailure, hemodialysis, uremia, toxicity, preeclampsia, okt3 therapy,anti-cd3 therapy, cytokine therapy, chemotherapy, radiation therapy(e.g., including but not limited toasthenia, anemia, cachexia, and thelike), chronic salicylate intoxication, and the like. See, e.g., theMerck Manual, 12th-17th Editions, Merck & Company, Rahway, N.J. (1972,1977, 1982, 1987, 1992, 1999), Pharmacotherapy Handbook, Wells et al.,eds., Second Edition, Appleton and Lange, Stamford, Conn. (1998, 2000),each entirely incorporated by reference.

[0152] The present invention also provides a method for modulating ortreating at least one cardiovascular disease in a cell, tissue, organ,animal, or patient, including, but not limited to, at least one ofcardiac stun syndrome, myocardial infarction, congestive heart failure,stroke, ischemic stroke, hemorrhage, arteriosclerosis, atherosclerosis,restenosis, diabetic ateriosclerotic disease, hypertension, arterialhypertension, renovascular hypertension, syncope, shock, syphilis of thecardiovascular system, heart failure, cor pulmonale, primary pulmonaryhypertension, cardiac arrhythmias, atrial ectopic beats, atrial flutter,atrial fibrillation (sustained or paroxysmal), post perfusion syndrome,cardiopulmonary bypass inflammation response, chaotic or multifocalatrial tachycardia, regular narrow QRS tachycardia, specific arrythmias,ventricular fibrillation, His bundle arrythmias, atrioventricular block,bundle branch block, myocardial ischemic disorders, coronary arterydisease, angina pectoris, myocardial infarction, cardiomyopathy, dilatedcongestive cardiomyopathy, restrictive cardiomyopathy, valvular heartdiseases, endocarditis, pericardial disease, cardiac tumors, aordic andperipheral aneuryisms, aortic dissection, inflammation of the aorta,occulsion of the abdominal aorta and its branches, peripheral vasculardisorders, occulsive arterial disorders, peripheral atherloscleroticdisease, thromboangitis obliterans, functional peripheral arterialdisorders, Raynaud's phenomenon and disease, acrocyanosis,erythromelalgia, venous diseases, venous thrombosis, varicose veins,arteriovenous fistula, lymphederma, lipedema, unstable angina,reperfusion injury, post pump syndrome, ischemia-reperfusion injury, andthe like. Such a method can optionally comprise administering aneffective amount of a composition or pharmaceutical compositioncomprising at least one anti-IL-12 antibody to a cell, tissue, organ,animal or patient in need of such modulation, treatment or therapy.

[0153] The present invention also provides a method for modulating ortreating at least one infectious disease in a cell, tissue, organ,animal or patient, including, but not limited to, at least one of: acuteor chronic bacterial infection, acute and chronic parasitic orinfectious processes, including bacterial, viral and fungal infections,HIV infection/HIV neuropathy, meningitis, hepatitis (A, B or C, or thelike), septic arthritis, peritonitis, pneumonia, epiglottitis, e. coli0157:h7, hemolytic uremic syndrome/thrombolytic thrombocytopenicpurpura, malaria, dengue hemorrhagic fever, leishmaniasis, leprosy,toxic shock syndrome, streptococcal myositis, gas gangrene,mycobacterium tuberculosis, mycobacterium avium intracellulare,pneumocystis carinii pneumonia, pelvic inflammatory disease,orchitis/epidydimitis, legionella, lyme disease, influenza a,epstein-barr virus, vital-associated hemaphagocytic syndrome, vitalencephalitis/aseptic meningitis, and the like;

[0154] The present invention also provides a method for modulating ortreating at least one malignant disease in a cell, tissue, organ, animalor patient, including, but not limited to, at least one of: leukemia,acute leukemia, acute lymphoblastic leukemia (ALL), B-cell, T-cell orFAB ALL, acute myeloid leukemia (AML), chromic myelocytic leukemia(CML), chronic lymphocytic leukemia (CLL), hairy cell leukemia,myelodyplastic syndrome (MDS), a lymphoma, Hodgkin's disease, amalignamt lymphoma, non-hodgkin's lymphoma, Burkitt's lymphoma, multiplemyeloma, Kaposi's sarcoma, colorectal carcinoma, pancreatic carcinoma,nasopharyngeal carcinoma, malignant histiocytosis, paraneoplasticsyndrome/hypercalcemia of malignancy, solid tumors, adenocarcinomas,sarcomas, malignant melanoma, hemangioma, metastatic disease, cancerrelated bone resorption, cancer related bone pain, and the like.

[0155] The present invention also provides a method for modulating ortreating at least one neurologic disease in a cell, tissue, organ,animal or patient, including, but not limited to, at least one of:neurodegenerative diseases, multiple sclerosis, migraine headache, AIDSdementia complex, demyelinating diseases, such as multiple sclerosis andacute transverse myelitis; extrapyramidal and cerebellar disorders' suchas lesions of the corticospinal system; disorders of the basal gangliaor cerebellar disorders; hyperkinetic movement disorders such asHuntington's Chorea and senile chorea; drug-induced movement disorders,such as those induced by drugs which block CNS dopamine receptors;hypokinetic movement disorders, such as Parkinson's disease; Progressivesupranucleo Palsy; structural lesions of the cerebellum; spinocerebellardegenerations, such as spinal ataxia, Friedreich's ataxia, cerebellarcortical degenerations, multiple systems degenerations (Mencel,Dejerine-Thomas, Shi-Drager, and Machado-Joseph); systemic disorders(Refsum's disease, abetalipoprotemia, ataxia, telangiectasia, andmitochondrial multi.system disorder); demyelinating core disorders, suchas multiple sclerosis, acute transverse myelitis; and disorders of themotor unit’ such as neurogenic muscular atrophies (anterior horn celldegeneration, such as amyotrophic lateral sclerosis, infantile spinalmuscular atrophy and juvenile spinal muscular atrophy); Alzheimer'sdisease; Down's Syndrome in middle age; Diffuse Lewy body disease;Senile Dementia of Lewy body type; Wernicke-Korsakoff syndrome; chronicalcoholism; Creutzfeldt-Jakob disease; Subacute sclerosingpanencephalitis, Hallerrorden-Spatz disease; and Dementia pugilistica,and the like. Such a method can optionally comprise administering aneffective amount of a composition or pharmaceutical compositioncomprising at least one TNF antibody or specified portion or variant toa cell, tissue, organ, animal or patient in need of such modulation,treatment or therapy. See, e.g., the Merck Manual, 16, Edition, Merck &Company, Rahway, N.J. (1992)

[0156] Any method of the present invention can comprise administering aneffective amount of a composition or pharmaceutical compositioncomprising at least one anti-IL-12 antibody to a cell, tissue, organ,animal or patient in need of such modulation, treatment or therapy. Sucha method can optionally further comprise co-administration orcombination therapy for treating such immune diseases, wherein theadministering of said at least one anti-IL-12 antibody, specifiedportion or variant thereof, further comprises administering, beforeconcurrently, and/or after, at least one selected from at least one TNFantagonist (e.g., but not limited to a TNF antibody or fragment, asoluble TNF receptor or fragment, fusion proteins thereof, or a smallmolecule TNF antagonist), an antirheumatic (e.g., methotrexate,auranofin, aurothioglucose, azathioprine, etanercept, gold sodiumthiomalate, hydroxychloroquine sulfate, leflunomide, sulfasalzine), amuscle relaxant, a narcotic, a non-steroid anti-inflammatory drug(NSAID), an analgesic, an anesthetic, a sedative, a local anethetic, aneuromuscular blocker, an antimicrobial (e.g., aminoglycoside, anantifungal, an antiparasitic, an antiviral, a carbapenem, cephalosporin,a flurorquinolone, a macrolide, a penicillin, a sulfonamide, atetracycline, another antimicrobial), an antipsoriatic, acorticosteriod, an anabolic steroid, a diabetes related agent, amineral, a nutritional, a thyroid agent, a vitamin, a calcium relatedhormone, an antidiarrheal, an antitussive, an antiemetic, an antiulcer,a laxative, an anticoagulant, an erythropieitin (e.g., epoetin alpha), afilgrastim (e.g., G-CSF, Neupogen), a sargramostim (GM-CSF, Leukine), animmunization, an immunoglobulin, an immunosuppressive (e.g.,basiliximab, cyclosporine, daclizumab), a growth hormone, a hormonereplacement drug, an estrogen receptor modulator, a mydriatic, acycloplegic, an alkylating agent, an antimetabolite, a mitoticinhibitor, a radiopharmaceutical, an antidepressant, antimanic agent, anantipsychotic, an anxiolytic, a hypnotic, a sympathomimetic, astimulant, donepezil, tacrine, an asthma medication, a beta agonist, aninhaled steroid, a leukotriene inhibitor, a methylxanthine, a cromolyn,an epinephrine or analog, domase alpha (Pulmozyme), a cytokine or acytokine antagonistm. Suitable dosages are well known in the art. See,e.g., Wells et al., eds., Pharmacotherapy Handbook, 2^(nd) Edition,Appleton and Lange, Stamford, Conn. (2000); PDR Pharmacopoeia, TarasconPocket Pharmacopoeia 2000, Deluxe Edition, Tarascon Publishing, LomaLinda, Calif. (2000), each of which references are entirely incorporatedherein by reference.

[0157] TNF antagonists suitable for compositions, combination therapy,co-administration, devices and/or methods of the present invention(further comprising at least one anti body, specified portion andvariant thereof, of the present invention), include, but are not limitedto, anti-TNF antibodies, antigen-binding fragments thereof, and receptormolecules which bind specifically to TNF; compounds which prevent and/orinhibit TNF synthesis, TNF release or its action on target cells, suchas thalidomide, tenidap, phosphodiesterase inhibitors (e.g,pentoxifylline and rolipram), A2b adenosine receptor agonists and A2badenosine receptor enhancers; compounds which prevent and/or inhibit TNFreceptor signalling, such as mitogen activated protein (MAP) kinaseinhibitors; compounds which block and/or inhibit membrane TNF cleavage,such as metalloproteinase inhibitors; compounds which block and/orinhibit TNF activity, such as angiotensin converting enzyme (ACE)inhibitors (e.g., captopril); and compounds which block and/or inhibitTNF production and/or synthesis, such as MAP kinase inhibitors.

[0158] As used herein, a “tumor necrosis factor antibody,” “TNFantibody,” “TNF antibody,” or fragment and the like decreases, blocks,inhibits, abrogates or interferes with TNF activity in vitro, in situand/or preferably in vivo. For example, a suitable TNF human antibody ofthe present invention can bind TNFα and includes anti-TNF antibodies,antigen-binding fragments thereof, and specified mutants or domainsthereof that bind specifically to TNFα. A suitable TNF anttibody orfragment can also decrease block, abrogate, interfere, prevent and/orinhibit TNF RNA, DNA or protein synthesis, TNF release, TNF receptorsignaling, membrane TNF cleavage, TNF activity, TNF production and/orsynthesis.

[0159] Chimeric antibody cA2 consists of the antigen binding variableregion of the high-affinity neutralizing mouse anti-human TNFα IgG1antibody, designated A2, and the constant regions of a human IgG1, kappaimmunoglobulin. The human IgG1 Fc region improves allogeneic antibodyeffector function, increases the circulating serum half-life anddecreases the immunogenicity of the antibody. The avidity and epitopespecificity of the chimeric antibody cA2 is derived from the variableregion of the murine antibody A2. In a particular embodiment, apreferred source for nucleic acids encoding the variable region of themurine antibody A2 is the A2 hybridoma cell line.

[0160] Chimeric A2 (cA2) neutralizes the cytotoxic effect of bothnatural and recombinant human TNFα in a dose dependent manner. Frombinding assays of chimeric antibody cA2 and recombinant human TNFα, theaffinity constant of chimeric antibody cA2 was calculated to be1.04×10¹⁰M⁻¹. Preferred methods for determining monoclonal antibodyspecificity and affinity by competitive inhibition can be found inHarlow, et al., antibodies: A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1988; Colligan et al., eds.,Current Protocols in Immunology, Greene Publishing Assoc. and WileyInterscience, New York, (1992-2000); Kozbor et al., Immunol. Today,4:72-79 (1983); Ausubel et al., eds. Current Protocols in MolecularBiology, Wiley Interscience, New York (1987-2000); and Muller, Meth.Enzymol., 92:589-601 (1983), which references are entirely incorporatedherein by reference.

[0161] In a particular embodiment, murine monoclonal antibody A2 isproduced by a cell line designated c134A. Chimeric antibody cA2 isproduced by a cell line designated c168A.

[0162] Additional examples of monoclonal anti-TNF antibodies that can beused in the present invention are described in the art (see, e.g., U.S.Pat. No. 5,231,024; Möller, A. et al., Cytokine 2(3):162-169 (1990);U.S. application Ser. No. 07/943,852 (filed Sep. 11, 1992); Rathjen etal., International Publication No. WO 91/02078 (published Feb. 21,1991); Rubin et al., EPO Patent Publication No. 0 218 868 (publishedApr. 22, 1987); Yone et al., EPO Patent Publication No. 0 288 088 (Oct.26, 1988); Liang, et al., Biochem. Biophys. Res. Comm. 137:847-854(1986); Meager, et al., Hybridoma 6:305-311 (1987); Fendly et al.,Hybridoma 6:359-369 (1987); Bringman, et al., Hybridoma 6:489-507(1987); and Hirai, et al., J. Immunol. Meth. 96:57-62 (1987), whichreferences are entirely incorporated herein by reference).

[0163] TNF Receptor Molecules

[0164] Preferred TNF receptor molecules useful in the present inventionare those that bind TNF with high affinity (see, e.g., Feldmann et al.,International Publication No. WO 92/07076 (published Apr. 30, 1992);Schall et al., Cell 61:361-370 (1990); and Loetscher et al., Cell61:351-359 (1990), which references are entirely incorporated herein byreference) and optionally possess low immunogenicity. In particular, the55 kDa (p55 TNF-R) and the 75 kDa (p75 TNF-R) TNF cell surface receptorsare useful in the present invention. Truncated forms of these receptors,comprising the extracellular domains (ECD) of the receptors orfunctional portions thereof (see, e.g., Corcoran et al., Eur. J.Biochem. 223:831-840 (1994)), are also useful in the present invention.Truncated forms of the TNF receptors, comprising the ECD, have beendetected in urine and serum as 30 kDa and 40 kDa TNF inhibitory bindingproteins (Engelmann, H. et al., J. Biol. Chem. 265:1531-1536 (1990)).TNF receptor multimeric molecules and TNF immunoreceptor fusionmolecules, and derivatives and fragments or portions thereof, areadditional examples of TNF receptor molecules which are useful in themethods and compositions of the present invention. The TNF receptormolecules which can be used in the invention are characterized by theirability to treat patients for extended periods with good to excellentalleviation of symptoms and low toxicity. Low immunogenicity and/or highaffinity, as well as other undefined properties, can contribute to thetherapeutic results achieved.

[0165] TNF receptor multimeric molecules useful in the present inventioncomprise all or a functional portion of the ECD of two or more TNFreceptors linked via one or more polypeptide linkers or other nonpeptidelinkers, such as polyethylene glycol (PEG). The multimeric molecules canfurther comprise a signal peptide of a secreted protein to directexpression of the multimeric molecule. These multimeric molecules andmethods for their production have been described in U.S. applicationSer. No. 08/437,533 (filed May 9, 1995), the content of which isentirely incorporated herein by reference.

[0166] TNF immunoreceptor fusion molecules useful in the methods andcompositions of the present invention comprise at least one portion ofone or more immunoglobulin molecules and all or a functional portion ofone or more TNF receptors. These immunoreceptor fusion molecules can beassembled as monomers, or hetero- or homo-multimers. The immunoreceptorfusion molecules can also be monovalent or multivalent. An example ofsuch a TNF immunoreceptor fusion molecule is TNF receptor/IgG fusionprotein. TNF immunoreceptor fusion molecules and methods for theirproduction have been described in the art (Lesslauer et al., Eur. J.Immunol. 21:2883-2886 (1991); Ashkenazi et al., Proc. Natl. Acad. Sci.USA 88:10535-10539 (1991); Peppel et al., J. Exp. Med. 174:1483-1489(1991); Kolls et al., Proc. Natl. Acad. Sci. USA 91:215-219 (1994);Butler et al., Cytokine 6(6):616-623 (1994); Baker et al., Eur. J.Immunol. 24:2040-2048 (1994); Beutler et al., U.S. Pat. No. 5,447,851;and U.S. application Ser. No. 08/442,133 (filed May 16, 1995), each ofwhich references are entirely incorporated herein by reference). Methodsfor producing immunoreceptor fusion molecules can also be found in Caponet al., U.S. Pat. No. 5,116,964; Capon et al., U.S. Pat. No. 5,225,538;and Capon et al., Nature 337:525-531 (1989), which references areentirely incorporated herein by reference.

[0167] A functional equivalent, derivative, fragment or region of TNFreceptor molecule refers to the portion of the TNF receptor molecule, orthe portion of the TNF receptor molecule sequence which encodes TNFreceptor molecule, that is of sufficient size and sequences tofunctionally resemble TNF receptor molecules that can be used in thepresent invention (e.g., bind TNF with high affinity and possess lowimmunogenicity). A functional equivalent of TNF receptor molecule alsoincludes modified TNF receptor molecules that functionally resemble TNFreceptor molecules that can be used in the present invention (e.g., bindTNF with high affinity and possess low immunogenicity). For example, afunctional equivalent of TNF receptor molecule can contain a “SILENT”codon or one or more amino acid substitutions, deletions or additions(e.g., substitution of one acidic amino acid for another acidic aminoacid; or substitution of one codon encoding the same or differenthydrophobic amino acid for another codon encoding a hydrophobic aminoacid). See Ausubel et al., Current Protocols in Molecular Biology,Greene Publishing Assoc. and Wiley-Interscience, New York (1987-2000).

[0168] Cytokines include any known cytokine. See, e.g.,www.CopewithCytokines.com. Cytokine antagonists include, but are notlimited to, any antibody, fragment or mimetic, any soluble receptor,fragment or mimetic, any small molecule antagonist, or any combinationthereof.

[0169] Therapeutic Treatments. Any method of the present invention cancomprise a method for treating a IL-12 mediated disorder, comprisingadministering an effective amount of a composition or pharmaceuticalcomposition comprising at least one anti-IL-12 antibody to a cell,tissue, organ, animal or patient in need of such modulation, treatmentor therapy. Such a method can optionally further compriseco-administration or combination therapy for treating such immunediseases, wherein the administering of said at least one anti-IL-12antibody, specified portion or variant thereof, further comprisesadministering, before concurrently, and/or after, at least one selectedfrom at least one of at least one selected from at least one TNFantagonist (e.g., but not limited to a TNF antibody or fragment, asoluble TNF receptor or fragment, fusion proteins thereof, or a smallmolecule TNF antagonist), an antirheumatic (e.g., methotrexate,auranofin, aurothioglucose, azathioprine, etanercept, gold sodiumthiomalate, hydroxychloroquine sulfate, leflunomide, sulfasalzine), amuscle relaxant, a narcotic, a non-steroid anti-inflammatory drug(NSAID), an analgesic, an anesthetic, a sedative, a local anethetic, aneuromuscular blocker, an antimicrobial (e.g., aminoglycoside, anantifungal, an antiparasitic, an antiviral, a carbapenem, cephalosporin,a flurorquinolone, a macrolide, a penicillin, a sulfonamide, atetracycline, another antimicrobial), an antipsoriatic, acorticosteriod, an anabolic steroid, a diabetes related agent, amineral, a nutritional, a thyroid agent, a vitamin, a calcium relatedhormone, an antidiarrheal, an antitussive, an antiemetic, an antiulcer,a laxative, an anticoagulant, an erythropieitin (e.g., epoetin alpha), afilgrastim (e.g., G-CSF, Neupogen), a sargramostim (GM-CSF, Leukine), animmunization, an immunoglobulin, an immunosuppressive (e.g.,basiliximab, cyclosporine, daclizumab), a growth hormone, a hormonereplacement drug, an estrogen receptor modulator, a mydriatic, acycloplegic, an alkylating agent, an antimetabolite, a mitoticinhibitor, a radiopharmaceutical, an antidepressant, antimanic agent, anantipsychotic, an anxiolytic, a hypnotic, a sympathomimetic, astimulant, donepezil, tacrine, an asthma medication, a beta agonist, aninhaled steroid, a leukotriene inhibitor, a methylxanthine, a cromolyn,an epinephrine or analog, dornase alpha (Pulmozyme), a cytokine or acytokine antagonist.

[0170] Typically, treatment of pathologic conditions is effected byadministering an effective amount or dosage of at least one anti-IL-12antibody composition that total, on average, a range from at least about0.01 to 500 milligrams of at least one anti-IL-12 antibody per kilogramof patient per dose, and preferably from at least about 0.1 to 100milligrams antibody/kilogram of patient per single or multipleadministration, depending upon the specific activity of contained in thecomposition. Alternatively, the effective serum concentration cancomprise 0.1-5000 μg/ml serum concentration per single or multipleadminstration. Suitable dosages are known to medical practitioners andwill, of course, depend upon the particular disease state, specificactivity of the composition being administered, and the particularpatient undergoing treatment. In some instances, to achieve the desiredtherapeutic amount, it can be necessary to provide for repeatedadministration, i.e., repeated individual administrations of aparticular monitored or metered dose, where the individualadministrations are repeated until the desired daily dose or effect isachieved.

[0171] Preferred doses can optionally include 0.1, 0.2, 0.3, 0.4, 0.5,0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 and/or 100-500mg/kg/administration, or any range, value or fraction thereof, or toachieve a serum concentration of 0.1, 0.5, 0.9, 1.0, 1.1, 1.2, 1.5, 1.9,2.0, 2.5, 2.9, 3.0, 3.5, 3.9, 4.0, 4.5, 4.9, 5.0, 5.5, 5.9, 6.0, 6.5,6.9, 7.0, 7.5, 7.9, 8.0, 8.5, 8.9, 9.0, 9.5, 9.9, 10, 10.5, 10.9, 11,11.5, 11.9, 20, 12.5, 12.9, 13.0, 13.5, 13.9, 14.0, 14.5, 4.9, 5.0,5.5., 5.9, 6.0, 6.5, 6.9, 7.0, 7.5, 7.9, 8.0, 8.5, 8.9, 9.0, 9.5, 9.9,10, 10.5, 10.9, 11, 11.5, 11.9, 12, 12.5, 12.9, 13.0, 13.5, 13.9, 14,14.5, 15, 15.5, 15.9, 16, 16.5, 16.9, 17, 17.5, 17.9, 18, 18.5, 18.9,19, 19.5, 19.9, 20, 20.5, 20.9, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 96, 100, 200, 300, 400,500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500,and/or 5000 μg/ml serum concentration per single or multipleadministration, or any range, value or fraction thereof.

[0172] Alternatively, the dosage administered can vary depending uponknown factors, such as the pharmacodynamic characteristics of theparticular agent, and its mode and route of administration; age, health,and weight of the recipient; nature and extent of symptoms, kind ofconcurrent treatment, frequency of treatment, and the effect desired.Usually a dosage of active ingredient can be about 0.1 to 100 milligramsper kilogram of body weight. Ordinarily 0.1 to 50, and preferably 0.1 to10 milligrams per kilogram per administration or in sustained releaseform is effective to obtain desired results.

[0173] As a non-limiting example, treatment of humans or animals can beprovided as a one-time or periodic dosage of at least one antibody ofthe present invention 0.1 to 100 mg/kg, such as 0.5, 0.9, 1.0, 1.1, 1.5,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100mg/kg, per day, on at least one of day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, or alternatively oradditionally, at least one of week 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, or 52, or alternatively or additionally, at least one of1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20years, or any combination thereof, using single, infusion or repeateddoses.

[0174] Dosage forms (composition) suitable for internal administrationgenerally contain from about 0.1 milligram to about 500 milligrams ofactive ingredient per unit or container. In these pharmaceuticalcompositions the active ingredient will ordinarily be present in anamount of about 0.5-99.999% by weight based on the total weight of thecomposition.

[0175] For parenteral administration, the antibody can be formulated asa solution, suspension, emulsion or lyophilized powder in association,or separately provided, with a pharmaceutically acceptable parenteralvehicle. Examples of such vehicles are water, saline, Ringer's solution,dextrose solution, and 1-10% human serum albumin. Liposomes andnonaqueous vehicles such as fixed oils can also be used. The vehicle orlyophilized powder can contain additives that maintain isotonicity(e.g., sodium chloride, mannitol) and chemical stability (e.g., buffersand preservatives). The formulation is sterilized by known or suitabletechniques.

[0176] Suitable pharmaceutical carriers are described in the most recentedition of Remington's Pharmaceutical Sciences, A. Osol, a standardreference text in this field.

[0177] Alternative Administration

[0178] Many known and developed modes of can be used according to thepresent invention for administering pharmaceutically effective amountsof at least one anti-IL-12 antibody according to the present invention.While pulmonary administration is used in the following description,other modes of administration can be used according to the presentinvention with suitable results.

[0179] IL-12 antibodies of the present invention can be delivered in acarrier, as a solution, emulsion, colloid, or suspension, or as a drypowder, using any of a variety of devices and methods suitable foradministration by inhalation or other modes described here within orknown in the art.

[0180] Parenteral Formulations and Administration

[0181] Formulations for parenteral administration can contain as commonexcipients sterile water or saline, polyalkylene glycols such aspolyethylene glycol, oils of vegetable origin, hydrogenated naphthalenesand the like. Aqueous or oily suspensions for injection can be preparedby using an appropriate emulsifier or humidifier and a suspending agent,according to known methods. Agents for injection can be a non-toxic,non-orally administrable diluting agent such as aquous solution or asterile injectable solution or suspension in a solvent. As the usablevehicle or solvent, water, Ringer's solution, isotonic saline, etc. areallowed; as an ordinary solvent, or suspending solvent, sterileinvolatile oil can be used. For these purposes, any kind of involatileoil and fatty acid can be used, including natural or synthetic orsemisynthetic fatty oils or fatty acids; natural or synthetic orsemisynthtetic mono- or di- or tri-glycerides. Parental administrationis known in the art and includes, but is not limited to, conventionalmeans of injections, a gas pressured needle-less injection device asdescribed in U.S. Pat. No. 5,851,198, and a laser perforator device asdescribed in U.S. Pat. No. 5,839,446 entirely incorporated herein byreference.

[0182] Alternative Delivery

[0183] The invention further relates to the administration of at leastone anti-IL-12 antibody by parenteral, subcutaneous, intramuscular,intravenous, intrarticular, intrabronchial, intraabdominal,intracapsular, intracartilaginous, intracavitary, intracelial,intracelebellar, intracerebroventricular, intracolic, intracervical,intragastric, intrahepatic, intramyocardial, intraosteal, intrapelvic,intrapericardiac, intraperitoneal, intrapleural, intraprostatic,intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal,intrasynovial, intrathoracic, intrauterine, intravesical, bolus,vaginal, rectal, buccal, sublingual, intranasal, or transdermal means.At least one anti-IL-12 antibody composition can be prepared for use forparenteral (subcutaneous, intramuscular or intravenous) or any otheradministration particularly in the form of liquid solutions orsuspensions; for use in vaginal or rectal administration particularly insemisolid forms such as, but not limited to, creams and suppositories;for buccal, or sublingual administration such as, but not limited to, inthe form of tablets or capsules; or intranasally such as, but notlimited to, the form of powders, nasal drops or aerosols or certainagents; or transdermally such as not limited to a gel, ointment, lotion,suspension or patch delivery system with chemical enhancers such asdimethyl sulfoxide to either modify the skin structure or to increasethe drug concentration in the transdermal patch (Junginger, et al. In“Drug Permeation Enhancement”; Hsieh, D. S., Eds., pp. 59-90 (MarcelDekker, Inc. New York 1994, entirely incorporated herein by reference),or with oxidizing agents that enable the application of formulationscontaining proteins and peptides onto the skin (WO 98/53847), orapplications of electric fields to create transient transport pathwayssuch as electroporation, or to increase the mobility of charged drugsthrough the skin such as iontophoresis, or application of ultrasoundsuch as sonophoresis (U.S. Pat. Nos. 4,309,989 and 4,767,402) (the abovepublications and patents being entirely incorporated herein byreference).

[0184] Pulmonary/Nasal Administration

[0185] For pulmonary administration, preferably at least one anti-IL-12antibody composition is delivered in a particle size effective forreaching the lower airways of the lung or sinuses. According to theinvention, at least one anti-IL-12 antibody can be delivered by any of avariety of inhalation or nasal devices known in the art foradministration of a therapeutic agent by inhalation. These devicescapable of depositing aerosolized formulations in the sinus cavity oralveoli of a patient include metered dose inhalers, nebulizers, drypowder generators, sprayers, and the like. Other devices suitable fordirecting the pulmonary or nasal administration of antibodies are alsoknown in the art. All such devices can use of formulations suitable forthe administration for the dispensing of antibody in an aerosol. Suchaerosols can be comprised of either solutions (both aqueous and nonaqueous) or solid particles. Metered dose inhalers like the Ventolin®metered dose inhaler, typically use a propellent gas and requireactuation during inspiration (See, e.g., WO 94/16970, WO 98/35888). Drypowder inhalers like Turbuhaler™ (Astra), Rotahaler® (Glaxo), Diskus®(Glaxo), Spiros™ inhaler (Dura), devices marketed by InhaleTherapeutics, and the Spinhaler® powder inhaler (Fisons), usebreath-actuation of a mixed powder (U.S. Pat. No. 4,668,218 Astra, EP237507 Astra, WO 97/25086 Glaxo, WO 94/08552 Dura, U.S. Pat. No.5,458,135 Inhale, WO 94/06498 Fisons, entirely incorporated herein byreference). Nebulizers like AERx™ Aradigm, the Ultravent® nebulizer(Mallinckrodt), and the Acorn II® nebulizer (Marquest Medical Products)(U.S. Pat. No. 5,404,871 Aradigm, WO 97/22376), the above referencesentirely incorporated herein by reference, produce aerosols fromsolutions, while metered dose inhalers, dry powder inhalers, etc.generate small particle aerosols. These specific examples ofcommercially available inhalation devices are intended to be arepresentative of specific devices suitable for the practice of thisinvention, and are not intended as limiting the scope of the invention.Preferably, a composition comprising at least one anti-IL-12 antibody isdelivered by a dry powder inhaler or a sprayer. There are a severaldesirable features of an inhalation device for administering at leastone antibody of the present invention. For example, delivery by theinhalation device is advantageously reliable, reproducible, andaccurate. The inhalation device can optionally deliver small dryparticles, e.g. less than about 10 μm, preferably about 1-5 μm, for goodrespirability.

[0186] Administration of IL-12 antibody Compositions as a Spray

[0187] A spray including IL-12 antibody composition protein can beproduced by forcing a suspension or solution of at least one anti-IL-12antibody through a nozzle under pressure. The nozzle size andconfiguration, the applied pressure, and the liquid feed rate can bechosen to achieve the desired output and particle size. An electrospraycan be produced, for example, by an electric field in connection with acapillary or nozzle feed. Advantageously, particles of at least oneanti-IL-12 antibody composition protein delivered by a sprayer have aparticle size less than about 10 μm, preferably in the range of about 1μm to about 5 μm, and most preferably about 2 μm to about 3 μm.

[0188] Formulations of at least one anti-IL-12 antibody compositionprotein suitable for use with a sprayer typically include antibodycomposition protein in an aqueous solution at a concentration of about0.1 mg to about 100 mg of at least one anti-IL-12 antibody compositionprotein per ml of solution or mg/gm, or any range or value therein,e.g., but not limited to, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or100 mg/ml or mg/gm. The formulation can include agents such as anexcipient, a buffer, an isotonicity agent, a preservative, a surfactant,and, preferably, zinc. The formulation can also include an excipient oragent for stabilization of the antibody composition protein, such as abuffer, a reducing agent, a bulk protein, or a carbohydrate. Bulkproteins useful in formulating antibody composition proteins includealbumin, protamine, or the like. Typical carbohydrates useful informulating antibody composition proteins include sucrose, mannitol,lactose, trehalose, glucose, or the like. The antibody compositionprotein formulation can also include a surfactant, which can reduce orprevent surface-induced aggregation of the antibody composition proteincaused by atomization of the solution in forming an aerosol. Variousconventional surfactants can be employed, such as polyoxyethylene fattyacid esters and alcohols, and polyoxyethylene sorbitol fatty acidesters. Amounts will generally range between 0.001 and 14% by weight ofthe formulation. Especially preferred surfactants for purposes of thisinvention are polyoxyethylene sorbitan monooleate, polysorbate 80,polysorbate 20, or the like. Additional agents known in the art forformulation of a protein such as IL-12 antibodies, or specified portionsor variants, can also be included in the formulation.

[0189] Administration of IL-12 Antibody Compositions by a Nebulizer

[0190] antibody composition protein can be administered by a nebulizer,such as jet nebulizer or an ultrasonic nebulizer. Typically, in a jetnebulizer, a compressed air source is used to create a high-velocity airjet through an orifice. As the gas expands beyond the nozzle, alow-pressure region is created, which draws a solution of antibodycomposition protein through a capillary tube connected to a liquidreservoir. The liquid stream from the capillary tube is sheared intounstable filaments and droplets as it exits the tube, creating theaerosol. A range of configurations, flow rates, and baffle types can beemployed to achieve the desired performance characteristics from a givenjet nebulizer. In an ultrasonic nebulizer, high-frequency electricalenergy is used to create vibrational, mechanical energy, typicallyemploying a piezoelectric transducer. This energy is transmitted to theformulation of antibody composition protein either directly or through acoupling fluid, creating an aerosol including the antibody compositionprotein. Advantageously, particles of antibody composition proteindelivered by a nebulizer have a particle size less than about 10 μm,preferably in the range of about 1 μm to about 5 μm, and most preferablyabout 2 μm to about 3 μm.

[0191] Formulations of at least one anti-IL-12 antibody suitable for usewith a nebulizer, either jet or ultrasonic, typically include aconcentration of about 0.1 mg to about 100 mg of at least one anti-IL-12antibody protein per ml of solution. The formulation can include agentssuch as an excipient, a buffer, an isotonicity agent, a preservative, asurfactant, and, preferably, zinc. The formulation can also include anexcipient or agent for stabilization of the at least one anti-IL-12antibody composition protein, such as a buffer, a reducing agent, a bulkprotein, or a carbohydrate. Bulk proteins useful in formulating at leastone anti-IL-12 antibody composition proteins include albumin, protamine,or the like. Typical carbohydrates useful in formulating at least oneanti-IL-12 antibody include sucrose, mannitol, lactose, trehalose,glucose, or the like. The at least one anti-IL-12 antibody formulationcan also include a surfactant, which can reduce or preventsurface-induced aggregation of the at least one anti-IL-12 antibodycaused by atomization of the solution in forming an aerosol. Variousconventional surfactants can be employed, such as polyoxyethylene fattyacid esters and alcohols, and polyoxyethylene sorbital fatty acidesters. Amounts will generally range between 0.001 and 4% by weight ofthe formulation. Especially preferred surfactants for purposes of thisinvention are polyoxyethylene sorbitan mono-oleate, polysorbate 80,polysorbate 20, or the like. Additional agents known in the art forformulation of a protein such as antibody protein can also be includedin the formulation.

[0192] Administration of IL-12 Antibody Compositions by a Metered DoseInhaler

[0193] In a metered dose inhaler (MDI), a propellant, at least oneanti-IL-12 antibody, and any excipients or other additives are containedin a canister as a mixture including a liquefied compressed gas.Actuation of the metering valve releases the mixture as an aerosol,preferably containing particles in the size range of less than about 10μm, preferably about 1 μm to about 5 μm, and most preferably about 2 μmto about 3 μm. The desired aerosol particle size can be obtained byemploying a formulation of antibody composition protein produced byvarious methods known to those of skill in the art, includingjet-milling, spray drying, critical point condensation, or the like.Preferred metered dose inhalers include those manufactured by 3M orGlaxo and employing a hydrofluorocarbon propellant.

[0194] Formulations of at least one anti-IL-12 antibody for use with ametered-dose inhaler device will generally include a finely dividedpowder containing at least one anti-IL-12 antibody as a suspension in anon-aqueous medium, for example, suspended in a propellant with the aidof a surfactant. The propellant can be any conventional materialemployed for this purpose, such as chlorofluorocarbon, ahydrochlorofluorocarbon, a hydrofluorocarbon, or a hydrocarbon,including trichlorofluoromethane, dichlorodifluoromethane,dichlorotetrafluoroethanol and 1,1,1,2-tetrafluoroethane, HFA-134a(hydrofluroalkane-134a), HFA-227 (hydrofluroalkane-227), or the like.Preferably the propellant is a hydrofluorocarbon. The surfactant can bechosen to stabilize the at least one anti-IL-12 antibody as a suspensionin the propellant, to protect the active agent against chemicaldegradation, and the like. Suitable surfactants include sorbitantrioleate, soya lecithin, oleic acid, or the like. In some casessolution aerosols are preferred using solvents such as ethanol.Additional agents known in the art for formulation of a protein such asprotein can also be included in the formulation.

[0195] One of ordinary skill in the art will recognize that the methodsof the current invention can be achieved by pulmonary administration ofat least one anti-IL-12 antibody compositions via devices not describedherein.

[0196] Oral Formulations and Administration

[0197] Formulations for oral rely on the co-administration of adjuvants(e.g., resorcinols and nonionic surfactants such as polyoxyethyleneoleyl ether and n-hexadecylpolyethylene ether) to increase artificiallythe permeability of the intestinal walls, as well as theco-administration of enzymatic inhibitors (e.g., pancreatic trypsininhibitors, diisopropylfluorophosphate (DFF) and trasylol) to inhibitenzymatic degradation. The active constituent compound of the solid-typedosage form for oral administration can be mixed with at least oneadditive, including sucrose, lactose, cellulose, mannitol, trehalose,raffinose, maltitol, dextran, starches, agar, arginates, chitins,chitosans, pectins, gum tragacanth, gum arabic, gelatin, collagen,casein, albumin, synthetic or semisynthetic polymer, and glyceride.These dosage forms can also contain other type(s) of additives, e.g.,inactive diluting agent, lubricant such as magnesium stearate, paraben,preserving agent such as sorbic acid, ascorbic acid, .alpha.-tocopherol,antioxidant such as cysteine, disintegrator, binder, thickener,buffering agent, sweetening agent, flavoring agent, perfuming agent,etc.

[0198] Tablets and pills can be further processed into enteric-coatedpreparations. The liquid preparations for oral administration includeemulsion, syrup, elixir, suspension and solution preparations allowablefor medical use. These preparations can contain inactive diluting agentsordinarily used in said field, e.g., water. Liposomes have also beendescribed as drug delivery systems for insulin and heparin (U.S. Pat.No. 4,239,754). More recently, microspheres of artificial polymers ofmixed amino acids (proteinoids) have been used to deliverpharmaceuticals (U.S. Pat. No. 4,925,673). Furthermore, carriercompounds described in U.S. Pat. No. 5,879,681 and U.S. Pat. No.5,5,871,753 are used to deliver biologically active agents orally areknown in the art.

[0199] Mucosal Formulations and Administration

[0200] For absorption through mucosal surfaces, compositions and methodsof administering at least one anti-IL-12 antibody include an emulsioncomprising a plurality of submicron particles, a mucoadhesivemacromolecule, a bioactive peptide, and an aqueous continuous phase,which promotes absorption through mucosal surfaces by achievingmucoadhesion of the emulsion particles (U.S. Pat. Nos. 5,514,670).Mucous surfaces suitable for application of the emulsions of the presentinvention can include corneal, conjunctival, buccal, sublingual, nasal,vaginal, pulmonary, stomachic, intestinal, and rectal routes ofadministration. Formulations for vaginal or rectal administration, e.g.suppositories, can contain as excipients, for example,polyalkyleneglycols, vaseline, cocoa butter, and the like. Formulationsfor intranasal administration can be solid and contain as excipients,for example, lactose or can be aqueous or oily solutions of nasal drops.For buccal administration excipients include sugars, calcium stearate,magnesium stearate, pregelinatined starch, and the like (U.S. Pat. No.5,849,695).

[0201] Transdermal Formulations and Administration

[0202] For transdermal administration, the at least one anti-IL-12antibody is encapsulated in a delivery device such as a liposome orpolymeric nanoparticles, microparticle, microcapsule, or microspheres(referred to collectively as microparticles unless otherwise stated). Anumber of suitable devices are known, including microparticles made ofsynthetic polymers such as polyhydroxy acids such as polylactic acid,polyglycolic acid and copolymers thereof, polyorthoesters,polyanhydrides, and polyphosphazenes, and natural polymers such ascollagen, polyamino acids, albumin and other proteins, alginate andother polysaccharides, and combinations thereof (U.S. Pat. No.5,814,599).

[0203] Prolonged Administration and Formulations

[0204] It can be sometimes desirable to deliver the compounds of thepresent invention to the subject over prolonged periods of time, forexample, for periods of one week to one year from a singleadministration. Various slow release, depot or implant dosage forms canbe utilized. For example, a dosage form can contain a pharmaceuticallyacceptable non-toxic salt of the compounds that has a low degree ofsolubility in body fluids, for example, (a) an acid addition salt with apolybasic acid such as phosphoric acid, sulfuric acid, citric acid,tartaric acid, tannic acid, pamoic acid, alginic acid, polyglutamicacid, naphthalene mono- or di-sulfonic acids, polygalacturonic acid, andthe like; (b) a salt with a polyvalent metal cation such as zinc,calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel,cadmium and the like, or with an organic cation formed from e.g.,N,N′-dibenzyl-ethylenediamine or ethylenediamine; or (c) combinations of(a) and (b) e.g. a zinc tannate salt. Additionally, the compounds of thepresent invention or, preferably, a relatively insoluble salt such asthose just described, can be formulated in a gel, for example, analuminum monostearate gel with, e.g. sesame oil, suitable for injection.Particularly preferred salts are zinc salts, zinc tannate salts, pamoatesalts, and the like. Another type of slow release depot formulation forinjection would contain the compound or salt dispersed for encapsulatedin a slow degrading, non-toxic, non-antigenic polymer such as apolylactic acid/polyglycolic acid polymer for example as described inU.S. Pat. No. 3,773,919. The compounds or, preferably, relativelyinsoluble salts such as those described above can also be formulated incholesterol matrix silastic pellets, particularly for use in animals.Additional slow release, depot or implant formulations, e.g. gas orliquid liposomes are known in the literature (U.S. Pat. No. 5,770,222and “Sustained and Controlled Release Drug Delivery Systems”, J. R.Robinson ed., Marcel Dekker, Inc., N.Y., 1978).

[0205] Having generally described the invention, the same will be morereadily understood by reference to the following examples, which areprovided by way of illustration and are not intended as limiting.

EXAMPLE 1 Cloning and Expression of IL-12 Antibody in Mammalian Cells

[0206] A typical mammalian expression vector contains at least onepromoter element, which mediates the initiation of transcription ofmRNA, the antibody coding sequence, and signals required for thetermination of transcription and polyadenylation of the transcript.Additional elements include enhancers, Kozak sequences and interveningsequences flanked by donor and acceptor sites for RNA splicing. Highlyefficient transcription can be achieved with the early and latepromoters from SV40, the long terminal repeats (LTRS) from Retroviruses,e.g., RSV, HTLVI, HIVI and the early promoter of the cytomegalovirus(CMV). However, cellular elements can also be used (e.g., the humanactin promoter). Suitable expression vectors for use in practicing thepresent invention include, for example, vectors such as pIRES1neo,pRetro-Off, pRetro-On, PLXSN, or pLNCX (Clonetech Labs, Palo Alto,Calif.), pcDNA3.1 (+/−), pcDNA/Zeo (+/−) or pcDNA3.1/Hygro (+/−)(Invitrogen), PSVL and PMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC37152), pSV2dhfr (ATCC 37146) and pBC12MI (ATCC 67109). Mammalian hostcells that could be used include human Hela 293, H9 and Jurkat cells,mouse NIH3T3 and C127 cells, Cos 1, Cos 7 and CV 1, quail QC1-3 cells,mouse L cells and Chinese hamster ovary (CHO) cells.

[0207] Alternatively, the gene can be expressed in stable cell linesthat contain the gene integrated into a chromosome. The co-transfectionwith a selectable marker such as dhfr, gpt, neomycin, or hygromycinallows the identification and isolation of the transfected cells.

[0208] The transfected gene can also be amplified to express largeamounts of the encoded antibody. The DHFR (dihydrofolate reductase)marker is useful to develop cell lines that carry several hundred oreven several thousand copies of the gene of interest. Another usefulselection marker is the enzyme glutamine synthase (GS) (Murphy, et al.,Biochem. J. 227:277-279 (1991); Bebbington, et al., Bio/Technology10:169-175 (1992)). Using these markers, the mammalian cells are grownin selective medium and the cells with the highest resistance areselected. These cell lines contain the amplified gene(s) integrated intoa chromosome. Chinese hamster ovary (CHO) and NSO cells are often usedfor the production of antibodies.

[0209] The expression vectors pC1 and pC4 contain the strong promoter(LTR) of the Rous Sarcoma Virus (Cullen, et al., Molec. Cell. Biol.5:438-447 (1985)) plus a fragment of the CMV-enhancer (Boshart, et al.,Cell 41:521-530 (1985)). Multiple cloning sites, e.g., with therestriction enzyme cleavage sites BamHI, XbaI and Asp718, facilitate thecloning of the gene of interest. The vectors contain in addition the 3′intron, the polyadenylation and termination signal of the ratpreproinsulin gene.

[0210] Cloning and Expression in CHO Cells

[0211] The vector pC4 is used for the expression of IL-12 antibody.Plasmid pC4 is a derivative of the plasmid pSV2-dhfr (ATCC Accession No.37146). The plasmid contains the mouse DHFR gene under control of theSV40 early promoter. Chinese hamster ovary- or other cells lackingdihydrofolate activity that are transfected with these plasmids can beselected by growing the cells in a selective medium (e.g., alpha minusMEM, Life Technologies, Gaithersburg, Md.) supplemented with thechemotherapeutic agent methotrexate. The amplification of the DHFR genesin cells resistant to methotrexate (MTX) has been well documented (see,e.g., F. W. Alt, et al., J. Biol. Chem. 253:1357-1370 (1978); J. L.Hamlin and C. Ma, Biochem. et Biophys. Acta 1097:107-143 (1990); and M.J. Page and M. A. Sydenham, Biotechnology 9:64-68 (1991)). Cells grownin increasing concentrations of MTX develop resistance to the drug byoverproducing the target enzyme, DHFR, as a result of amplification ofthe DHFR gene. If a second gene is linked to the DHFR gene, it isusually co-amplified and over-expressed. It is known in the art thatthis approach can be used to develop cell lines carrying more than 1,000copies of the amplified gene(s). Subsequently, when the methotrexate iswithdrawn, cell lines are obtained that contain the amplified geneintegrated into one or more chromosome(s) of the host cell.

[0212] Plasmid pC4 contains for expressing the gene of interest thestrong promoter of the long terminal repeat (LTR) of the Rous SarcomaVirus (Cullen, et al., Molec. Cell. Biol. 5:438-447 (1985)) plus afragment isolated from the enhancer of the immediate early gene of humancytomegalovirus (CMV) (Boshart, et al., Cell 41:521-530 (1985)).Downstream of the promoter are BamHI, XbaI, and Asp718 restrictionenzyme cleavage sites that allow integration of the genes. Behind thesecloning sites the plasmid contains the 3′ intron and polyadenylationsite of the rat preproinsulin gene. Other high efficiency promoters canalso be used for the expression, e.g., the human b-actin promoter, theSV40 early or late promoters or the long terminal repeats from otherretroviruses, e.g., HIV and HTLVI. Clontech's Tet-Off and Tet-On geneexpression systems and similar systems can be used to express the IL-12in a regulated way in mammalian cells (M. Gossen, and H. Bujard, Proc.Natl. Acad. Sci. USA 89: 5547-5551 (1992)). For the polyadenylation ofthe mRNA other signals, e.g., from the human growth hormone or globingenes can be used as well. Stable cell lines carrying a gene of interestintegrated into the chromosomes can also be selected uponco-transfection with a selectable marker such as gpt, G418 orhygromycin. It is advantageous to use more than one selectable marker inthe beginning, e.g., G418 plus methotrexate.

[0213] The plasmid pC4 is digested with restriction enzymes and thendephosphorylated using calf intestinal phosphatase by procedures knownin the art. The vector is then isolated from a 1% agarose gel.

[0214] The DNA sequence encoding the complete IL-12 antibody is used,e.g., as presented in SEQ ID NOS:INSERT MAB AA SEQ ID 1, and INSERT MABAA SEQ ID NO2, corresponding to HC and LC variable regions of a IL-12antibody of the present invention, according to known method steps.Isolated nucleic acid encoding a suitable human constant region (i.e.,HC and LC regions) is also used in this construct (e.g., as provided invector p1351: INSERT ATCC ACCESSION NUMBER AND ADDITIONAL HC/LCplasmids)

[0215] The isolated variable and constant region encoding DNA and thedephosphorylated vector are then ligated with T4 DNA ligase. E. coli HB101 or XL-1 Blue cells are then transformed and bacteria are identifiedthat contain the fragment inserted into plasmid pC4 using, for instance,restriction enzyme analysis.

[0216] Chinese hamster ovary (CHO) cells lacking an active DHFR gene areused for transfection. 5 g of the expression plasmid pC4 iscotransfected with 0.5 g of the plasmid pSV2-neo using lipofectin. Theplasmid pSV2neo contains a dominant selectable marker, the neo gene fromTn5 encoding an enzyme that confers resistance to a group of antibioticsincluding G418. The cells are seeded in alpha minus MEM supplementedwith 1 g/ml G418. After 2 days, the cells are trypsinized and seeded inhybridoma cloning plates (Greiner, Germany) in alpha minus MEMsupplemented with 10, 25, or 50 ng/ml of methotrexate plus 1 g/ml G418.After about 10-14 days single clones are trypsinized and then seeded in6-well petri dishes or 10 ml flasks using different concentrations ofmethotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM). Clones growing atthe highest concentrations of methotrexate are then transferred to new6-well plates containing even higher concentrations of methotrexate (1mM, 2 mM, 5 mM, 10 mM, 20 mM). The same procedure is repeated untilclones are obtained that grow at a concentration of 100-200 mM.Expression of the desired gene product is analyzed, for instance, bySDS-PAGE and Western blot or by reverse phase HPLC analysis.

EXAMPLE 2 Generation of High Affinity Human IgG Monoclonal AntibodiesReactive with Human IL-12 Using Transgenic Mice

[0217] Summary

[0218] Transgenic mice have been used that contain human heavy and lightchain immunoglobulin genes to generate high affinity, completely human,monoclonal antibodies that can be used therapeutically to inhibit theaction of IL-12 for the treatment of one or more IL-12-mediated disease.(CBA/J×C57/BL6/J) F₂ hybrid mice containing human variable and constantregion antibody transgenes for both heavy and light chains are immunizedwith human recombinant IL-12 (Taylor et al., Intl. Immunol. 6:579-591(1993); Lonberg, et al., Nature 368:856-859 (1994); Neuberger, M.,Nature Biotech. 14:826 (1996); Fishwild, et al., Nature Biotechnology14:845-851 (1996)). Several fusions yielded one or more panels ofcompletely human IL-12 reactive IgG monoclonal antibodies. Thecompletely human anti-IL-12 antibodies are further characterized. Allare IgG1. Such antibodies are found to have affinity constants somewherebetween 1×10⁹ and 9×10¹². The unexpectedly high affinities of thesefully human monoclonal antibodies make them suitable candidates fortherapeutic applications in IL-12 related diseases, pathologies ordisorders.

[0219] Abbreviations

[0220] BSA—bovine serum albumin

[0221] CO₂—carbon dioxide

[0222] DMSO—dimethyl sulfoxide

[0223] EIA—enzyme immunoassay

[0224] FBS—fetal bovine serum

[0225] H₂O₂—hydrogen peroxide

[0226] HRP—horseradish peroxidase\

[0227] ID—interadermal

[0228] Ig—immunoglobulin

[0229] IL-12—interleukin-12

[0230] IP—intraperitoneal

[0231] IV—intravenous

[0232] Mab—monoclonal antibody

[0233] OD—optical density

[0234] OPD—o-Phenylenediamine dihydrochloride

[0235] PEG—polyethylene glycol

[0236] PSA—penicillin, streptomycin, amphotericin

[0237] RT—room temperature

[0238] SQ—subcutaneous

[0239] v/v—volume per volume

[0240] w/v—weight per volume

[0241] Materials and Methods

[0242] Animals

[0243] Transgenic mice that can express human antibodies are known inthe art (and are commecially available (e.g., from GenPharmInternational, San Jose, Calif.; Abgenix, Freemont, Calif., and others)that express human immunoglobulins but not mouse IgM or Ig. For example,such transgenic mice contain human sequence transgenes that undergoV(D)J joining, heavy-chain class switching, and somatic mutation togenerate a repertoire of human sequence immunoglobulins (Lonberg, etal., Nature 368:856-859 (1994)). The light chain transgene can bederived, e.g., in part from a yeast artificial chromosome clone thatincludes nearly half of the germline human V region. In addition, theheavy-chain transgene can encode both human μ and human 1 (Fishwild, etal., Nature Biotechnology 14:845-851 (1996)) and/or 3 constant regions.Mice derived from appropriate genotopic lineages can be used in theimmunization and fusion processes to generate fully human monoclonalantibodies to IL-12.

[0244] Immunization

[0245] One or more immunization schedules can be used to generate theanti-IL-12 human hybridomas. The first several fusions can be performedafter the following exemplary immunization protocol, but other similarknown protocols can be used. Several 14-20 week old female and/orsurgically castrated transgenic male mice are immunized IP and/or IDwith 1-1000 μg of recombinant human IL-12 emulsified with an equalvolume of TITERMAX or complete Freund's adjuvant in a final volume of100-400 μL (e.g., 200). Each mouse can also optionally receive 1-10 μgin 100 μL physiological saline at each of 2 SQ sites. The mice can thenbe immunized 1-7, 5-12, 10-18, 17-25 and/or 21-34 days later IP (1-400μg) and SQ (1-400 μg×2) with IL-12 emulsified with an equal volume ofTITERMAX or incomplete Freund's adjuvant. Mice can be bled 12-25 and25-40 days later by retro-orbital puncture without anti-coagulant. Theblood is then allowed to clot at RT for one hour and the serum iscollected and titered using an IL-12 EIA assay according to knownmethods. Fusions are performed when repeated injections do not causetiters to increase. At that time, the mice can be given a final IVbooster injection of 1-400 μg IL-12 diluted in 100 μL physiologicalsaline. Three days later, the mice can be euthanized by cervicaldislocation and the spleens removed aseptically and immersed in 10 mL ofcold phosphate buffered saline (PBS) containing 100 U/mL penicillin, 100μg/mL streptomycin, and 0.25 μg/mL amphotericin B (PSA). The splenocytesare harvested by sterilely perfusing the spleen with PSA-PBS. The cellsare washed once in cold PSA-PBS, counted using Trypan blue dye exclusionand resuspended in RPMI 1640 media containing 25 mM Hepes.

[0246] Cell Fusion

[0247] Fusion can be carried out at a 1:1 to 1:10 ratio of murinemyeloma cells to viable spleen cells according to known methods, e.g.,as known in the art. As a non-limiting example, spleen cells and myelomacells can be pelleted together. The pellet can then be slowlyresuspended, over 30 seconds, in 1 mL of 50% (w/v) PEG/PBS solution (PEGmolecular weight 1,450, Sigma) at 37 C. The fusion can then be stoppedby slowly adding 10.5 mL of RPMI 1640 medium containing 25 mM Hepes (37C) over 1 minute. The fused cells are centrifuged for 5 minutes at500-1500 rpm. The cells are then resuspended in HAT medium (RPMI 1640medium containing 25 mM Hepes, 10% Fetal Clone I serum (Hyclone), 1 mMsodium pyruvate, 4 mM L-glutamine, 10 μg/mL gentamicin, 2.5% Origenculturing supplement (Fisher), 10% 653-conditioned RPMI 1640/Hepesmedia, 50 μM 2-mercaptoethanol, 100 μM hypoxanthine, 0.4 μM aminopterin,and 16 μM thymidine) and then plated at 200 μL/well in fifteen 96-wellflat bottom tissue culture plates. The plates are then placed in ahumidified 37 C incubator containing 5% CO₂ and 95% air for 7-10 days.

[0248] Detection of Human IgG Anti-IL-12 Antibodies in Mouse Serum

[0249] Solid phase EIA's can be used to screen mouse sera for human IgGantibodies specific for human IL-12. Briefly, plates can be coated withIL-12 at 2 μg/mL in PBS overnight. After washing in 0.15M salinecontaining 0.02% (v/v) Tween 20, the wells can be blocked with 1% (w/v)BSA in PBS, 200 μL/well for 1 hour at RT. Plates are used immediately orfrozen at −20 C for future use. Mouse serum dilutions are incubated onthe IL-12 coated plates at 50 μL/well at RT for 1 hour. The plates arewashed and then probed with 50 μL/well HRP-labeled goat anti-human IgG,Fc specific diluted 1:30,000 in 1% BSA-PBS for 1 hour at RT. The platescan again be washed and 100 μL/well of the citrate-phosphate substratesolution (0. IM citric acid and 0.2M sodium phosphate, 0.01% H₂O₂ and 1mg/mL OPD) is added for 15 minutes at RT. Stop solution (4N sulfuricacid) is then added at 25 μL/well and the OD's are read at 490 nm via anautomated plate spectrophotometer.

[0250] Detection of Completely Human Immunoglobulins in HybridomaSupernates

[0251] Growth positive hybridomas secreting fully human immunoglobulinscan be detected using a suitable EIA. Briefly, 96 well pop-out plates(VWR, 610744) can be coated with 10 μg/mL goat anti-human IgG Fc insodium carbonate buffer overnight at 4 C. The plates are washed andblocked with 1% BSA-PBS for one hour at 37° C. and used immediately orfrozen at −20 C. Undiluted hybridoma supernatants are incubated on theplates for one hour at 37° C. The plates are washed and probed with HRPlabeled goat anti-human kappa diluted 1:10,000 in 1% BSA-PBS for onehour at 37° C. The plates are then incubated with substrate solution asdescribed above.

[0252] Determination of Fully Human Anti-IL-12 Reactivity

[0253] Hybridomas, as above, can be simultaneously assayed forreactivity to IL-12 using a suitable RIA or other assay. For example,supernatants are incubated on goat anti-human IgG Fc plates as above,washed and then probed with radiolabled IL-12 with appropriate countsper well for 1 hour at RT. The wells are washed twice with PBS and boundradiolabled IL-12 is quantitated using a suitable counter.

[0254] Human IgG1 anti-IL-12 secreting hybridomas can be expanded incell culture and serially subcloned by limiting dilution. The resultingclonal populations can be expanded and cryopreserved in freezing medium(95% FBS, 5% DMSO) and stored in liquid nitrogen.

[0255] Isotyping

[0256] Isotype determination of the antibodies can be accomplished usingan EIA in a format similar to that used to screen the mouse immune serafor specific titers. IL-12 can be coated on 96-well plates as describedabove and purified antibody at 2 μg/mL can be incubated on the plate forone hour at RT. The plate is washed and probed with HRP labeled goatanti-human IgG₁ or HRP labeled goat anti-human IgG₃ diluted at 1:4000 in1% BSA-PBS for one hour at RT. The plate is again washed and incubatedwith substrate solution as described above.

[0257] Binding Kinetics of Human Anti-Human IL-12 Antibodies with HumanIL-12

[0258] Binding characteristics for antibodies can be suitably assessedusing an IL-12 capture EIA and BIAcore technology, for example. Gradedconcentrations of purified human IL-12 antibodies can be assessed forbinding to EIA plates coated with 2 μg/mL of IL-12 in assays asdescribed above. The OD's can be then presented as semi-log plotsshowing relative binding efficiencies.

[0259] Quantitative binding constants can be obtained, e.g., as follows,or by any other known suitable method. A BIAcore CM-5 (carboxymethyl)chip is placed in a BIAcore 2000 unit. HBS buffer (0.01 M HEPES, 0.15 MNaCl, 3 mM EDTA, 0.005% v/v P20 surfactant, pH 7.4) is flowed over aflow cell of the chip at 5 μL/minute until a stable baseline isobtained. A solution (100 μL) of 15 mg of EDC(N-ethyl-N′-(3-dimethyl-aminopropyl)-carbodiimide hydrochloride) in 200μL water is added to 100 μL of a solution of 2.3 mg of NHS(N-hydroxysuccinimide) in 200 μL water. Forty (40) μL of the resultingsolution is injected onto the chip. Six μL of a solution of human IL-12(15 μg/mL in 10 mM sodium acetate, pH 4.8) is injected onto the chip,resulting in an increase of ca. 500 RU. The buffer is changed toTBS/Ca/Mg/BSA running buffer (20 mM Tris, 0.15 M sodium chloride, 2 mMcalcium chloride, 2 mM magnesium acetate, 0.5% Triton X-100, 25 μg/mLBSA, pH 7.4) and flowed over the chip overnight to equilibrate it and tohydrolyze or cap any unreacted succinimide esters.

[0260] Antibodies are dissolved in the running buffer at 33.33, 16.67,8.33, and 4.17 nM. The flow rate is adjusted to 30 μL/min and theinstrument temperature to 25 C. Two flow cells are used for the kineticruns, one on which IL-12 had been immobilized (sample) and a second,underivatized flow cell (blank). 120 μL of each antibody concentrationis injected over the flow cells at 30 μL/min (association phase)followed by an uninterrupted 360 seconds of buffer flow (dissociationphase). The surface of the chip is regenerated (interleukin-12/antibodycomplex dissociated) by two sequential injections of 30 μL each of 2 Mguanidine thiocyanate.

[0261] Analysis of the data is done using BIA evaluation 3.0 or CLAMP2.0, as known in the art. For each antibody concentration the blanksensogram is subtracted from the sample sensogram. A global fit is donefor both dissociation (k/_(d,) sec⁻¹) and association (k_(a), mol⁻¹sec⁻¹) and the dissociation constant (K_(D), mol) calculated(k_(d)/k_(a)). Where the antibody affinity is high enough that the RUsof antibody captured are >100, additional dilutions of the antibody arerun.

[0262] Results and Discussion

[0263] Generation of Anti-Human IL-12 Monoclonal Antibodies

[0264] Several fusions are performed and each fusion is seeded in 15plates (1440 wells/fusion) that yield several dozen antibodies specificfor human IL-12. Of these, some are found to consist of a combination ofhuman and mouse Ig chains. The remaining hybridomas secret anti-IL-12antibodies consisting solely of human heavy and light chains. Of thehuman hybridomas all are expected to be IgG1.

[0265] Binding Kinetics of Human Anti-Human IL-12 Antibodies

[0266] ELISA analysis confirms that purified antibody from most or allof these hybridomas bind IL-12 in a concentration-dependent manner.FIGS. 1-2 show the results of the relative binding efficiency of theseantibodies. In this case, the avidity of the antibody for its cognateantigen (epitope) is measured. It should be noted that binding IL-12directly to the EIA plate can cause denaturation of the protein and theapparent binding affinities cannot be reflective of binding toundenatured protein. Fifty percent binding is found over a range ofconcentrations.

[0267] Quantitative binding constants are obtained using BIAcoreanalysis of the human antibodies and reveals that several of the humanmonoclonal antibodies are very high affinity with K_(D) in the range of1×10⁻⁹ to 7×10^(−12.)

[0268] Conclusions

[0269] Several fusions are performed utilizing splenocytes from hybridmice containing human variable and constant region antibody transgenesthat are immunized with human IL-12. A set of several completely humanIL-12 reactive IgG monoclonal antibodies of the IgG1 isotype aregenerated. The completely human anti-IL-12 antibodies are furthercharacterized. Several of generated antibodies have affinity constantsbetween 1×10⁹ and 9×10¹². The unexpectedly high affinities of thesefully human monoclonal antibodies make them suitable for therapeuticapplications in IL-12-dependent diseases, pathologies or relatedconditions.

EXAMPLE 3 C340 is a Neutralizing Human Monoclonal Antibody

[0270] The bioactivity of IL-12 was shown to be neutralized by C340 in avariety of IL-12 dependent activity assays. Since IL-12 enhances IFNGAMMA production by NK cells and T lymphocytes, the effect of C340antibody on the upregulation of IFN GAMMA mRNA and the effect of C340 onthe production of IFN GAMMA protein was examined (Trinchieri, G.,Current Opinion in Immunology, 9:17-23 (1997), Morris, S.C., et al.,Journal of Immunology, 152:1047-1056 (1994)). The ability of C340 toneutralize IL-12 driven induction of lymphokine activated killer (LAK)cell activity was also investigated in these studies (Kutza, J. andMurasko, D. M., Mechanisms of Ageing and Development, 90:209-222 (1996),Stem, A. S., et al., Proceedings of the National Academy of Sciences ofthe U.S.A., 87:6808-6812 (1990)). Lastly, the effect of C340 onIL-12-mediated upregulation of CD95 cell surface expression on T and NKcells was tested (Medvedev, A. E., et al., Cytokine, 9:394-404 (1997)).

[0271] Inhibition of IFN Gamma mRNA Transcription

[0272] To determine whether C340 inhibits IL-12/IL-2 induced IFN GAMMAgene transcription in human PBL, a reverse transcription-PCR assay wasperformed. Specific primers for β-actin (a control for mRNA integrityand content) and IFN GAMMA were used to amplify the cDNA obtained fromstimulated human PBL. FIG. 3 shows C340 down regulates IFN GAMMA mRNA inIL-12/IL-2 activated (2 hour) PBMC.

[0273] Inhibition of Intracellular IFN GAMMA as Measured by FlowCytometry

[0274] In response to various signals and as a measure of activation, Tcells and NK cells can be induced to secrete cytokines. Morespecifically, PBL treated with IL-2 and IL-12 initiate substantialsynthesis of IFN gamma within 4-8 hours after stimulation. Thisproduction can be detected in the cytoplasm of Brefeldin-A treated PBLby flow cytometry. FIG. 4 demonstrates a 60% reduction in IFN GAMMAproduction in such cultures when C340 IL-12 was added in conjunctionwith IL-12 for five hours.

[0275] Inhibition of IL-12 Induced IFN GAMMA Secretion

[0276]FIG. 5 clearly shows that two different lots of C340 inhibited thesecretion of IFN GAMMA by peripheral blood lymphocytes in adose-dependent fashion. Four hundred picograms of IL-12 were premixedwith varying amounts of C340 and then added to IL-2 stimulated culturesof PBL's. When IFN GAMMA was measured by EIA after an 18-24 hourincubation, markedly diminished amounts of IFN GAMMA were detected withas little as 1 ?g/mL of C340 antibody.

[0277] Inhibition of IL-12 Induced LAK Cell Cytotoxicity

[0278] Raji cells, an IL-12 sensitive Burkitt lymphoma derived cellline, is an NK cell resistant, LAK cell sensitive cell line. Raji cells,in triplicate, were cultured for four hours with LAK cells which hadbeen activated with 400 pg/mL IL-12 and 10 U/mL IL-2 in the presence orabsence of the human monoclonal antibody C340 (5000 ng/mL or 50 ng/mL).FIG. 6 shows the results from three normal, healthy donors. IL-12+IL-2activation of effector cells resulted in an increasing cytotoxicactivity over that of cells activated with IL-2 alone. The C340 antibodyinhibited this IL-12 dependent effect. The magnitude of inhibition wasrelated to antibody concentration, with the highest concentration testedreducing cytotoxicity to background levels.

[0279] Inhibition of CD95 Upregulation

[0280] Reports have described IL-12-induced upregulation of CD95 on thesurface of highly purified CD56+ PBL. As can be seen in FIGS. 7A and 7B,distributional flow cytometric analysis revealed that CD95 expressionwas significantly upregulated on CD3+ T cells and CD56+ NK cells aftertreatment with IL-12 plus IL-2 for 72 hours. Concomitant anti-IL-12treatment inhibited CD95 expression in both CD3+ and CD56+ populations.CD3+ cells were inhibited by 50% (FIG. 7A), whereas CD56+ cells wereinhibited by ˜85% (FIG. 7B), as evidenced by a diminished MFI index(percent greater then unstimulated control).

EXAMPLE 4 Gene Cloning and Characterization

[0281] Genomic DNA fragments containing either the C340 heavy chain geneor the C340 light chain were cloned and purified. Genomic DNA purifiedfrom C340 hybridoma cells was partially digested with Sau3A restrictionenzyme and size-selected by centrifugal fractionation through a 10-40%sucrose gradient. DNA fragments in the size range of 15-23 kb werecloned into the bacteriophage vector, EMBL3, [commercially available ?]and packaged into phage particles. Several packaging reactions resultedin a library of 1 million bacteriophage clones. Approximately 600,000clones from the library were screened by plaque hybridization using32P-labeled genomic DNA fragments that contained either human IgG1 heavychain constant region sequences or human kappa light chain constantregion sequences as probe. Thirteen heavy chain and nine light chainclones were detected. Of these, three heavy chain clones and four lightchain clones were purified by two more rounds of screening. One of theheavy chain clones and two of the light chain clones were shown tocontain the 5′ and 3′ ends of the coding sequences by PCR analysis ofbacteriophage DNA. The DNA insert in heavy chain (HC) clone H4 was 16 kbin size and includes 3.6 kb of 5′ flanking and at least 2 kb of 3′flanking sequence. The DNA insert in light chain (LC) clone LC 1 was 15kb in size and included 4.4 kb of 5′ flanking and 6.0 kb of 3′ flankingsequence. The complete inserts were removed from the bacteriophagevector as SalI fragments and cloned between the XhoI and SalI sites ofplasmid expression vector p1351, which provided a gpt selectable markergene. Because there was an internal SalI site in the heavy chainvariable region coding sequence, two SalI fragments had to betransferred from bacteriophage H4 to the p1351 expression vector. Theresulting heavy and light chain expression plasmids were termed p1560and p1558, respectively. The orientations of the heavy and light chaingenes in these two plasmids relative to the p1351 vector sequences weredetermined using restriction enzyme analysis and PCR, respectively. Inboth cases, the orientations were such that the 5′ end of the Ab genefragment was proximal to the 3′ end of the gpt gene. Both strands of thecoding regions of the cloned genes were sequenced. The sequences ofplasmids p1560 and p1558 are presented in FIGS. 11A-11K and FIGS.13A-13J, respectively.

EXAMPLE 5 Preparation of Recombinant Cell Lines

[0282] Heavy chain plasmid p1560 was linearized by digestion with PvuIrestriction enzyme and light chain plasmid p1558 was linearized usingSalI restriction enzyme. p3×63Ag8.653 (653) and SP2/0-Ag14 (SP2/0) cellswere separately transfected with the premixed linearized plasmids byelectroporation and cells cultured and transfectants selected usingmycophenolic acid as described (Knight, et al., Molecular Immunology30:1443 (1993)). Cell supernatants from mycophenolic acid-resistantcolonies were assayed approximately two weeks later for human IgG (i.e.,recombinant C340 (rC340)). For this, cell supernatants were incubated on96-well ELISA plates that were coated with goat antibodies specific forthe Fc portion of human IgG. Human IgG which bound to the coated platewas detected using alkaline phosphatase-conjugated goat anti-human IgG(heavy chain+light chain) antibody and alkaline phosphatase substratesas described (Knight, et al., Molecular Immunology 30:1443 (1993)).Cells of the higher producing clones were transferred to 24-well culturedishes in standard media and expanded (IMDM, 5% FBS, 2 mM glutamine,mycophenolic acid selection mix). The amount of antibody produced (i.e.,secreated into the media of spent cultures) was carefully quantified byELISA using purified C340 mAb as the standard. Selected clones were thenexpanded in T75 flasks and the production of human IgG by these cloneswas quantified by ELISA. Based on these values, six independent 653transfectants and three independent SP2/0 transfectants were subcloned(by seeding an average of one cell per well in 96 well plates), thequantity of antibody produced by the subclones was determined byassaying (ELISA) supernatants from individual subclone colonies. Threesubclones, 653 transfectant 19-20 (C379B) and the SP2/0 transfectants84-81 (C381A) and 22-56 (C389A), were selected for further analysis.

[0283] Assay for rC340 Antigen Binding.

[0284] Prior to subcloning selected cell lines as described above, cellsupernatants from three parental lines (653 transfectants clone 2 andclone 18 and SP2/0 transfectant clone 1) were used to test the antigenbinding characteristics of rC340. The concentrations of rC340 in thethree cell supernatant samples were first determined by ELISA. Titratingamounts of the supernatant samples, or purified C340 positive control,were then incubated in 96-well plates coated with 2 μg/ml of humanIL-12. Bound mAb was then detected with alkaline phosphatase-conjugatedgoat anti-human IgG (heavy chain+light chain) antibody and theappropriate alkaline phosphatase substrates. As shown in FIG. 8, rC340bound specifically to human IL-12 in a manner indistinguishable from theoriginal C340 mAb.

[0285] Characterization of Selected Cell Lines.

[0286] Growth curve analyses were performed on C379B, C381A, and C389Aby seeding T75 flasks with a starting cell density of 2×10⁵ cells/ml instandard media or SFM-5 serum-free media and then monitoring cell numberand rC340 concentration on a daily basis until the cultures were spent.The results of cultures in standard media are shown in FIGS. 9A-9C.Maximal C340 mAb production levels for C379B, C381A, and C389A were 135μg/ml, 150 μg/ml, and 110 μg/ml, respectively. Attempts to adapt C379Bcells to SFM-5 media were not successful. C381A cells produced the sameamount of rC340 in SFM-5 media as in standard media, whereas C389A cellsproduced only half as much rC340 in SFM-5 media as in standard media.

[0287] The stability of rC340 mAb production over time for the threesubclones was assessed by culturing cells in 24-well dishes withstandard media or standard media without mycophenolic acid selection forvarying periods of time. Lines C379B and C381A were observed to stablyproduce rC340 in the presence or absence of selection for a period of 30days (the maximum time tested) and 75 days, respectively. Line C389A wasunstable and after 43 days of culture produced just 20% as much antibodyas at the beginning of the study.

[0288] It will be clear that the invention can be practiced otherwisethan as particularly described in the foregoing description andexamples.

[0289] Numerous modifications and variations of the present inventionare possible in light of the above teachings and, therefore, are withinthe scope of the appended claims.

1 15 1 5 PRT Homo sapiens 1 Thr Tyr Trp Leu Gly 1 5 2 17 PRT Homosapiens 2 Ile Met Ser Pro Val Asp Ser Asp Ile Arg Tyr Ser Pro Ser PheGln 1 5 10 15 Gly 3 10 PRT Homo sapiens 3 Pro Arg Pro Gly Gln Gly TyrPhe Asp Phe 1 5 10 4 11 PRT Homo sapiens 4 Arg Ala Ser Gln Gly Ile SerSer Trp Leu Ala 1 5 10 5 7 PRT Homo sapiens 5 Ala Ala Ser Ser Leu GlnSer 1 5 6 9 PRT Homo sapiens 6 Gln Gln Tyr Asn Ile Tyr Pro Tyr Thr 1 5 7119 PRT Homo sapiens 7 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val LysLys Pro Gly Glu 1 5 10 15 Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly TyrSer Phe Thr Thr Tyr 20 25 30 Trp Leu Gly Trp Val Arg Gln Met Pro Gly LysGly Leu Asp Trp Ile 35 40 45 Gly Ile Met Ser Pro Val Asp Ser Asp Ile ArgTyr Ser Pro Ser Phe 50 55 60 Gln Gly Gln Val Thr Met Ser Val Asp Lys SerIle Thr Thr Ala Tyr 65 70 75 80 Leu Gln Trp Asn Ser Leu Lys Ala Ser AspThr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Arg Arg Pro Gly Gln Gly Tyr PheAsp Phe Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 8108 PRT Homo sapiens 8 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu SerAla Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser GlnGly Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys AlaPro Lys Ser Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val ProSer Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr IleSer Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln GlnTyr Asn Ile Tyr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu IleLys Arg 100 105 9 503 PRT Homo sapiens 9 Arg Asn Leu Pro Val Ala Thr ProAsp Pro Gly Met Phe Pro Cys Leu 1 5 10 15 His His Ser Gln Asn Leu LeuArg Ala Val Ser Asn Met Leu Gln Lys 20 25 30 Ala Arg Gln Thr Leu Glu PheTyr Pro Cys Thr Ser Glu Glu Ile Asp 35 40 45 His Glu Asp Ile Thr Lys AspLys Thr Ser Thr Val Glu Ala Cys Leu 50 55 60 Pro Leu Glu Leu Thr Lys AsnGlu Ser Cys Leu Asn Ser Arg Glu Thr 65 70 75 80 Ser Phe Ile Thr Asn GlySer Cys Leu Ala Ser Arg Lys Thr Ser Phe 85 90 95 Met Met Ala Leu Cys LeuSer Ser Ile Tyr Glu Asp Leu Lys Met Tyr 100 105 110 Gln Val Glu Phe LysThr Met Asn Ala Lys Leu Leu Met Asp Pro Lys 115 120 125 Arg Gln Ile PheLeu Asp Gln Asn Met Leu Ala Val Ile Asp Glu Leu 130 135 140 Met Gln AlaLeu Asn Phe Asn Ser Glu Thr Val Pro Gln Lys Ser Ser 145 150 155 160 LeuGlu Glu Pro Asp Phe Tyr Lys Thr Lys Ile Lys Leu Cys Ile Leu 165 170 175Leu His Ala Phe Arg Ile Arg Ala Val Thr Ile Asp Arg Val Met Ser 180 185190 Tyr Leu Asn Ala Ser Ile Trp Glu Leu Lys Lys Asp Val Tyr Val Val 195200 205 Glu Leu Asp Trp Tyr Pro Asp Ala Pro Gly Glu Met Val Val Leu Thr210 215 220 Cys Asp Thr Pro Glu Glu Asp Gly Ile Thr Trp Thr Leu Asp GlnSer 225 230 235 240 Ser Glu Val Leu Gly Ser Gly Lys Thr Leu Thr Ile GlnVal Lys Glu 245 250 255 Phe Gly Asp Ala Gly Gln Tyr Thr Cys His Lys GlyGly Glu Val Leu 260 265 270 Ser His Ser Leu Leu Leu Leu His Lys Lys GluAsp Gly Ile Trp Ser 275 280 285 Thr Asp Ile Leu Lys Asp Gln Lys Glu ProLys Asn Lys Thr Phe Leu 290 295 300 Arg Cys Glu Ala Lys Asn Tyr Ser GlyArg Phe Thr Cys Trp Trp Leu 305 310 315 320 Thr Thr Ile Ser Thr Asp LeuThr Phe Ser Val Lys Ser Ser Arg Gly 325 330 335 Ser Ser Asp Pro Gln GlyVal Thr Cys Gly Ala Ala Thr Leu Ser Ala 340 345 350 Glu Arg Val Arg GlyAsp Asn Lys Glu Tyr Glu Tyr Ser Val Glu Cys 355 360 365 Gln Glu Asp SerAla Cys Pro Ala Ala Glu Glu Ser Leu Pro Ile Glu 370 375 380 Val Met ValAsp Ala Val His Lys Leu Lys Tyr Glu Asn Tyr Thr Ser 385 390 395 400 SerPhe Phe Ile Arg Asp Ile Ile Lys Pro Asp Pro Pro Lys Asn Leu 405 410 415Gln Leu Lys Pro Leu Lys Asn Ser Arg Gln Val Glu Val Ser Trp Glu 420 425430 Tyr Pro Asp Thr Trp Ser Thr Pro His Ser Tyr Phe Ser Leu Thr Phe 435440 445 Cys Val Gln Val Gln Gly Lys Ser Lys Arg Glu Lys Lys Asp Arg Val450 455 460 Phe Thr Asp Lys Thr Ser Ala Thr Val Ile Cys Arg Lys Asn AlaSer 465 470 475 480 Ile Ser Val Arg Ala Gln Asp Arg Tyr Tyr Ser Ser SerTrp Ser Glu 485 490 495 Trp Ala Ser Val Pro Cys Ser 500 10 15 DNA Homosapiens 10 agatatacta tgcac 15 11 51 DNA Homo sapiens 11 gttatatcatttgatggaag caataaatac tacgtagact ccgtgaaggg c 51 12 30 DNA Homo sapiens12 gaggcccggg gatcgtatgc ttttgatatc 30 13 42 DNA Homo sapiens 13ctctcctgca gggccagtca gagtgttagc agctacttag cc 42 14 18 DNA Homo sapiens14 gatgcatcca acagggcc 18 15 21 DNA Homo sapiens 15 cagcagcgtagcaactggcc t 21

What is claimed is:
 1. At least one isolated mammalian anti-IL-12antibody, comprising at least one variable region comprising SEQ ID NO:7or
 8. 2. An IL-12 antibody according to claim 1, wherein said antibodybinds IL-12 with an affinity of at least one selected from at least 10⁻⁹M, at least 10⁻¹⁰ M, at least 10 ⁻¹¹ M, or at least 10⁻¹² M.
 3. An IL-12antibody according to claim 1, wherein said antibody substantiallyneutralizes at least one activity of at least one IL-12 protein.
 4. Anisolated nucleic acid encoding at least one isolated mammaliananti-IL-12 antibody having at least one variable region comprising SEQID NO:7 or
 8. 5. An isolated nucleic acid vector comprising an isolatednucleic acid according to claim
 4. 6. A prokaryotic or eukaryotic hostcell comprising an isolated nucleic acid according to claim
 5. 7. A hostcell according to claim 6, wherein said host cell is at least oneselected from COS-1, COS-7, HEK293, BHK21, CHO, BSC-1, Hep G2, 653,SP2/0, 293, HeLa, myeloma, or lymphoma cells, or any derivative,immortalized or transformed cell thereof.
 8. A method for producing atleast one anti-IL-12 antibody, comprising translating a nucleic acidaccording to claim 4 under conditions in vitro, in vivo or in situ, suchthat the IL-12 antibody is expressed in detectable or recoverableamounts.
 9. A composition comprising at least one isolated mammaliananti-IL-12 antibody having at least one variable region comprising SEQID NO:7 or 8, and at least one pharmaceutically acceptable carrier ordiluent.
 10. A composition according to claim 9, further comprising atleast one composition comprising an effective amount of at least onecompound or protein selected from at least one of a detectable label orreporter, a TNF antagonist, an antirheumatic, a muscle relaxant, anarcotic, a non-steroid anti-inflammatory drug (NSAID), an analgesic, ananesthetic, a sedative, a local anethetic, a neuromuscular blocker, anantimicrobial, an antipsoriatic, a corticosteriod, an anabolic steroid,an erythropoietin, an immunization, an immunoglobulin, animmunosuppressive, a growth hormone, a hormone replacement drug, aradiopharmaceutical, an antidepressant, an antipsychotic, a stimulant,an asthma medication, a beta agonist, an inhaled steroid, an epinephrineor analog, a cytokine, or a cytokine antagonist.
 11. An anti-idiotypeantibody or fragment that specifically binds at least one isolatedmammalian anti-IL-12 antibody having at least one variable regioncomprising SEQ ID NO:7 or
 8. 12. A method for diagnosing or treating aIL-12 related condition in a cell, tissue, organ or animal, comprising(a) contacting or administering a composition comprising an effectiveamount of at least one isolated mammalian anti-IL-12 antibody having atleast one variable region comprising SEQ ID NO:7 or 8, with, or to, saidcell, tissue, organ or animal.
 13. A method according to claim 12,wherein said effective amount is 0.001-50 mg/kilogram of said cells,tissue, organ or animal.
 14. A method according to claim 12, whereinsaid contacting or said administrating is by at least one mode selectedfrom parenteral, subcutaneous, intramuscular, intravenous,intrarticular, intrabronchial, intraabdominal, intracapsular,intracartilaginous, intracavitary, intracelial, intracelebellar,intracerebroventricular, intracolic, intracervical, intragastric,intrahepatic, intramyocardial, intraosteal, intrapelvic,intrapericardiac, intraperitoneal, intrapleural, intraprostatic,intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal,intrasynovial, intrathoracic, intrauterine, intravesical, bolus,vaginal, rectal, buccal, sublingual, intranasal, or transdermal.
 15. Amethod according to claim 12, further comprising administering, prior,concurrently or after said (a) contacting or administering, at least onecomposition comprising an effective amount of at least one compound orprotein selected from at least one of a detectable label or reporter, aTNF antagonist, an antirheumatic, a muscle relaxant, a narcotic, anon-steroid anti-inflammatory drug (NSAID), an analgesic, an anesthetic,a sedative, a local anethetic, a neuromuscular blocker, anantimicrobial, an antipsoriatic, a corticosteriod, an anabolic steroid,an erythropoietin, an immunization, an immunoglobulin, animmunosuppressive, a growth hormone, a hormone replacement drug, aradiopharmaceutical, an antidepressant, an antipsychotic, a stimulant,an asthma medication, a beta agonist, an inhaled steroid, an epinephrineor analog, a cytokine, or a cytokine antagonist.
 16. A method accordingto claim 12, wherein said IL-12 related condition is psoriasis.
 17. Amethod according to claim 12, wherein said IL-12 related condition ismultiple sclerosis.
 18. A medical device, comprising at least oneisolated mammalian anti-IL-12 antibody having at least one variableregion comprising SEQ ID NO:7 or 8, wherein said device is suitable tocontacting or administerting said at least one anti-IL-12 antibody by atleast one mode selected from parenteral, subcutaneous, intramuscular,intravenous, intrarticular, intrabronchial, intraabdominal,intracapsular, intracartilaginous, intracavitary, intracelial,intracelebellar, intracerebroventricular, intracolic, intracervical,intragastric, intrahepatic, intramyocardial, intraosteal, intrapelvic,intrapericardiac, intraperitoneal, intrapleural, intraprostatic,intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal,intrasynovial, intrathoracic, intrauterine, intravesical, bolus,vaginal, rectal, buccal, sublingual, intranasal, or transdermal.
 19. Amethod for producing at least one isolated mammalian anti-IL-12 antibodyhaving at least one variable region comprising SEQ ID NO:7 or 8,comprising providing a host cell or transgenic animal or transgenicplant or plant cell capable of expressing in recoverable amounts saidantibody.
 20. At least one anti-IL-12 antibody produced by a methodaccording to claim
 19. 21. At least one isolated mammalian anti-IL-12antibody, comprising either (i) all of the heavy chain complementaritydetermining regions (CDR) amino acid sequences of SEQ ID NOS: 7, 8, and9; or (ii) all of the light chain CDR amino acids sequences of SEQ IDNOS: 10, 11, and
 12. 22. An IL-12 antibody according to claim 21,wherein said antibody binds IL-12 with an affinity of at least oneselected from at least 10⁻⁹ M, at least 10⁻¹⁰ M, at least 10⁻¹¹ M, or atleast 10⁻¹² M.
 23. An IL-12 antibody according to claim 21, wherein saidantibody substantially neutralizes at least one activity of at least oneIL-12 protein.
 24. An isolated nucleic acid encoding at least oneisolated mammalian anti-IL-12 antibody either (i) all of the heavy chainCDR amino acid sequences of SEQ ID NOS: 7, 8, and 9; or (ii) all of thelight chain CDR amino acids sequences of SEQ ID NOS: 10, 11, and
 12. 25.An isolated nucleic acid vector comprising an isolated nucleic acidaccording to claim
 4. 26. A prokaryotic or eukaryotic host cellcomprising an isolated nucleic acid according to claim
 25. 27. A hostcell according to claim 26, wherein said host cell is at least oneselected from COS-1, COS-7, HEK293, BK21, CHO, BSC-1, Hep G2, 653,SP2/0, 293, HeLa, myeloma, or lymphoma cells, or any derivative,immortalized or transformed cell thereof.
 28. A method for producing atleast one anti-IL-12 antibody, comprising translating a nucleic acidaccording to claim 24 under conditions in vitro, in vivo or in situ,such that the IL-12 antibody is expressed in detectable or recoverableamounts.
 29. A composition comprising at least one isolated mammaliananti-IL-12 antibody having either (i) all of the heavy chain CDR aminoacid sequences of SEQ ID NOS: 7, 8, and 9; or (ii) all of the lightchain CDR amino acids sequences of SEQ ID NOS: 10, 11, and 12, and atleast one pharmaceutically acceptable carrier or diluent.
 30. Acomposition according to claim 29, further comprising at least onecomposition comprising an effective amount of at least one compound orprotein selected from at least one of a detectable label or reporter, aTNF antagonist, an antirheumatic, a muscle relaxant, a narcotic, anon-steroid anti-inflammatory drug (NSAID), an analgesic, an anesthetic,a sedative, a local anethetic, a neuromuscular blocker, anantimicrobial, an antipsoriatic, a corticosteriod, an anabolic steroid,an erythropoietin, an immunization, an immunoglobulin, animmunosuppressive, a growth hormone, a hormone replacement drug, aradiopharmaceutical, an antidepressant, an antipsychotic, a stimulant,an asthma medication, a beta agonist, an inhaled steroid, an epinephrineor analog, a cytokine, or a cytokine antagonist.
 31. An anti-idiotypeantibody or fragment that specifically binds at least one isolatedmammalian anti-IL-12 antibody having either (i) all of the heavy chainCDR amino acid sequences of SEQ ID NOS: 7, 8, and 9; or (ii) all of thelight chain CDR amino acids sequences of SEQ ID NOS: 10, 11, and
 12. 32.A method for diagnosing or treating a IL-12 related condition in a cell,tissue, organ or animal, comprising (a) contacting or administering acomposition comprising an effective amount of at least one isolatedmammalian anti-IL-12 antibody having either (i) all of the heavy chainCDR amino acid sequences of SEQ ID NOS: 7, 8, and 9; or (ii) all of thelight chain CDR amino acids sequences of SEQ ID NOS: 10, 11, and 12,with, or to, said cell, tissue, organ or animal.
 33. A method accordingto claim 32, wherein said effective amount is 0.001-50 mg/kilogram ofsaid cells, tissue, organ or animal.
 34. A method according to claim 32,wherein said contacting or said administrating is by at least one modeselected from parenteral, subcutaneous, intramuscular, intravenous,intrarticular, intrabronchial, intraabdominal, intracapsular,intracartilaginous, intracavitary, intracelial, intracelebellar,intracerebroventricular, intracolic, intracervical, intragastric,intrahepatic, intramyocardial, intraosteal, intrapelvic,intrapericardiac, intraperitoneal, intrapleural, intraprostatic,intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal,intrasynovial, intrathoracic, intrauterine, intravesical, bolus,vaginal, rectal, buccal, sublingual, intranasal, or transdermal.
 35. Amethod according to claim 32, further comprising administering, prior,concurrently or after said (a) contacting or administering, at least onecomposition comprising an effective amount of at least one compound orprotein selected from at least one of a detectable label or reporter, aINF antagonist, an antirheumatic, a muscle relaxant, a narcotic, anon-steroid anti-inflammatory drug (NSAID), an analgesic, an anesthetic,a sedative, a local anethetic, a neuromuscular blocker, anantimicrobial, an antipsoriatic, a corticosteriod, an anabolic steroid,an erythropoietin, an immunization, an immunoglobulin, animmunosuppressive, a growth hormone, a hormone replacement drug, aradiopharmaceutical, an antidepressant, an antipsychotic, a stimulant,an asthma medication, a beta agonist, an inhaled steroid, an epinephrineor analog, a cytokine, or a cytokine antagonist.
 36. A method accordingto claim 32, wherein said IL-12 related condition is psoriasis.
 37. Amethod according to claim 32, wherein said IL-12 related condition ismultiple sclerosis.
 38. A medical device, comprising at least oneisolated mammalian anti-IL-12 antibody having either (i) all of theheavy chain CDR amino acid sequences of SEQ ID NOS: 7, 8, and 9; or (ii)all of the light chain CDR amino acids sequences of SEQ ID NOS: 10, 11,and 12, wherein said device is suitable to contacting or administertingsaid at least one anti-IL-12 antibody by at least one mode selected fromparenteral, subcutaneous, intramuscular, intravenous, intrarticular,intrabronchial, intraabdominal, intracapsular, intracartilaginous,intracavitary, intracelial, intracelebellar, intracerebroventricular,intracolic, intracervical, intragastric, intrahepatic, intramyocardial,intraosteal, intrapelvic, intrapericardiac, intraperitoneal,intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal,intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine,intravesical, bolus, vaginal, rectal, buccal, sublingual, intranasal, ortransdermal.
 39. A method for producing at least one isolated mammaliananti-IL-12 antibody having either (i) all of the heavy chain CDR aminoacid sequences of SEQ ID NOS: 7, 8, and 9; or (ii) all of the lightchain CDR amino acids sequences of SEQ ID NOS: 10, 11, and 12,comprising providing a host cell or transgenic animal or transgenicplant or plant cell capable of expressing in recoverable amounts saidantibody.
 40. At least one anti-IL-12 antibody produced by a methodaccording to claim
 39. 41. At least one isolated mammalian anti-IL-12antibody, comprising at least one heavy chain or light chain CDR havingthe amino acid sequence of at least one of SEQ ID NOS: 1, 2, 3, 4, 5, or6.
 42. An IL-12 antibody according to claim 41, wherein said antibodybinds IL-12 with an affinity of at least one selected from at least 10⁻⁹M, at least 10⁻¹⁰ M, at least 10 ⁻¹¹ M, or at least 10⁻¹² M.
 43. AnIL-12 antibody according to claim 41, wherein said antibodysubstantially neutralizes at least one activity of at least one IL-12protein.
 44. An isolated nucleic acid encoding at least one isolatedmammalian anti-IL-12 antibody having at least one heavy chain or lightchain CDR having the amino acid sequence of at least one of SEQ ID NOS:1, 2, 3, 4, 5, or
 6. 45. An isolated nucleic acid vector comprising anisolated nucleic acid according to claim
 44. 46. A prokaryotic oreukaryotic host cell comprising an isolated nucleic acid according toclaim
 45. 47. A host cell according to claim 46, wherein said host cellis at least one selected from COS-1, COS-7, HEK293, BHK21, CHO, BSC-1,Hep G2, 653, SP2/0, 293, HeLa, myeloma, or lymphoma cells, or anyderivative, immortalized or transformed cell thereof.
 48. A method forproducing at least one anti-IL-12 antibody, comprising translating anucleic acid according to claim 44 under conditions in vitro, in vivo orin situ, such that the IL-12 antibody is expressed in detectable orrecoverable amounts.
 49. A composition comprising at least one isolatedmammalian anti-IL-12 antibody having at least one heavy chain or lightchain CDR having the amino acid sequence of at least one of SEQ ID NOS:1, 2, 3, 4, 5, or 6, and at least one pharmaceutically acceptablecarrier or diluent.
 50. A composition according to claim 49, furthercomprising at least one composition comprising an effective amount of atleast one compound or protein selected from at least one of a detectablelabel or reporter, a TNF antagonist, an antirheumatic, a musclerelaxant, a narcotic, a non-steroid anti-inflammatory drug (NSA/D), ananalgesic, an anesthetic, a sedative, a local anethetic, a neuromuscularblocker, an antimicrobial, an antipsoriatic, a corticosteriod, ananabolic steroid, an erythropoietin, an immunization, an immunoglobulin,an immunosuppressive, a growth hormone, a hormone replacement drug, aradiopharmaceutical, an antidepressant, an antipsychotic, a stimulant,an asthma medication, a beta agonist, an inhaled steroid, an epinephrineor analog, a cytokine, or a cytokine antagonist.
 51. An anti-idiotypeantibody or fragment that specifically binds at least one isolatedmammalian anti-IL-12 antibody having at least one heavy chain or lightchain CDR having the amino acid sequence of at least one of SEQ ID NOS:1, 2, 3, 4, 5, or
 6. 52. A method for diagnosing or treating a IL-12related condition in a cell, tissue, organ or animal, comprising (a)contacting or administering a composition comprising an effective amountof at least one isolated mammalian anti-IL-12 antibody having at leastone heavy chain or light chain CDR having the amino acid sequence of atleast one of SEQ ID NOS: 1, 2, 3, 4, 5, or 6, with, or to, said cell,tissue, organ or animal.
 53. A method according to claim 52, whereinsaid effective amount is 0.001-50 mg/kilogram of said cells, tissue,organ or animal.
 54. A method according to claim 52, wherein saidcontacting or said administrating is by at least one mode selected fromparenteral, subcutaneous, intramuscular, intravenous, intrarticular,intrabronchial, intraabdominal, intracapsular, intracartilaginous,intracavitary, intracelial, intracelebellar, intracerebroventricular,intracolic, intracervical, intragastric, intrahepatic, intramyocardial,intraosteal, intrapelvic, intrapericardiac, intraperitoneal,intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal,intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine,intravesical, bolus, vaginal, rectal, buccal, sublingual, intranasal, ortransdermal.
 55. A method according to claim 52, further comprisingadministering, prior, concurrently or after said (a) contacting oradministering, at least one composition comprising an effective amountof at least one compound or protein selected from at least one of adetectable label or reporter, a TNF antagonist, an antirheumatic, amuscle relaxant, a narcotic, a non-steroid anti-inflammatory drug(NSAID), an analgesic, an anesthetic, a sedative, a local anethetic, aneuromuscular blocker, an antimicrobial, an antipsoriatic, acorticosteriod, an anabolic steroid, an erythropoietin, an immunization,an immunoglobulin, an immunosuppressive, a growth hormone, a hormonereplacement drug, a radiopharmaceutical, an antidepressant, anantipsychotic, a stimulant, an asthma medication, a beta agonist, aninhaled steroid, an epinephrine or analog, a cytokine, or a cytokineantagonist.
 56. A method according to claim 52, wherein said IL-12related condition is psoriasis.
 57. A method according to claim 52,wherein said IL-12 related condition is multiple sclerosis.
 58. Amedical device, comprising at least one isolated mammalian anti-IL-12antibody having at least one heavy chain or light chain CDR having theamino acid sequence of at least one of SEQ ID NOS: 1, 2, 3, 4, 5, or 6,wherein said device is suitable to contacting or administerting said atleast one anti-IL-12 antibody by at least one mode selected fromparenteral, subcutaneous, intramuscular, intravenous, intrarticular,intrabronchial, intraabdominal, intracapsular, intracartilaginous,intracavitary, intracelial, intracelebellar, intracerebroventricular,intracolic, intracervical, intragastric, intrahepatic, intramyocardial,intraosteal, intrapelvic, intrapericardiac, intraperitoneal,intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal,intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine,intravesical, bolus, vaginal, rectal, buccal, sublingual, intranasal, ortransdermal.
 59. A method for producing at least one isolated mammaliananti-IL-12 antibody having at least one heavy chain or light chain CDRhaving the amino acid sequence of at least one of SEQ ID NOS: 1, 2, 3,4, 5, or 6, comprising providing a host cell or transgenic animal ortransgenic plant or plant cell capable of expressing in recoverableamounts said antibody.
 60. At least one anti-IL-12 antibody produced bya method according to claim
 59. 61. At least one isolated mammaliananti-IL-12 antibody that binds to the same region of a IL-12 protein asan antibody comprising at least one heavy chain or light chain CDRhaving the amino acid sequence of at least one of SEQ ID NOS: 1, 2, 3,4, 5, or
 6. 62. An IL-12 antibody according to claim 61, wherein saidantibody binds IL-12 with an affinity of at least one selected from atleast 10⁻⁹ M, at least 10⁻¹⁰ M, at least 10⁻¹¹ M, or at least 10⁻¹² M.63. An IL-12 antibody according to claim 61, wherein said antibodysubstantially neutralizes at least one activity of at least one IL-12protein.
 64. An isolated nucleic acid encoding at least one isolatedmammalian anti-IL-12 antibody that binds to the same region of a IL-12protein as an antibody comprising at least one heavy chain or lightchain CDR having the amino acid sequence of at least one of SEQ ID NOS:1, 2, 3, 4, 5, or
 6. 65. An isolated nucleic acid vector comprising anisolated nucleic acid according to claim
 64. 66. A prokaryotic oreukaryotic host cell comprising an isolated nucleic acid according toclaim
 65. 67. A host cell according to claim 66, wherein said host cellis at least one selected from COS-1, COS-7, HEK293, BHK21, CHO, BSC-1,Hep G2, 653, SP2/0, 293, HeLa, myeloma, or lymphoma cells, or anyderivative, immortalized or transformed cell thereof.
 68. A method forproducing at least one anti-IL-12 antibody, comprising translating anucleic acid according to claim 64 under conditions in vitro, in vivo orin situ, such that the IL-12 antibody is expressed in detectable orrecoverable amounts.
 69. A composition comprising at least one isolatedmammalian anti-IL-12 antibody that binds to the same region of a IL-12protein as an antibody comprising at least one heavy chain or lightchain CDR having the amino acid sequence of at least one of SEQ ID NOS:1, 2, 3, 4, 5, or 6, and at least one pharmaceutically acceptablecarrier or diluent.
 70. A composition according to claim 69, furthercomprising at least one composition comprising an effective amount of atleast one compound or protein selected from at least one of a detectablelabel or reporter, a TNF antagonist, an antirheumatic, a musclerelaxant, a narcotic, a non-steroid anti-inflammatory drug (NSAID), ananalgesic, an anesthetic, a sedative, a local anethetic, a neuromuscularblocker, an antimicrobial, an antipsoriatic, a corticosteriod, ananabolic steroid, an erythropoietin, an immunization, an immunoglobulin,an immunosuppressive, a growth hormone, a hormone replacement drug, aradiopharmaceutical, an antidepressant, an antipsychotic, a stimulant,an asthma medication, a beta agonist, an inhaled steroid, an epinephrineor analog, a cytokine, or a cytokine antagonist.
 71. An anti-idiotypeantibody or fragment that specifically binds at least one isolatedmammalian anti-IL-12 antibody that binds to the same region of a IL-12protein as an antibody comprising at least one heavy chain or lightchain CDR having the amino acid sequence of at least one of SEQ ID NOS:1, 2, 3, 4, 5, or
 6. 72. A method for diagnosing or treating a IL-12related condition in a cell, tissue, organ or animal, comprising (a)contacting or administering a composition comprising an effective amountof at least one isolated mammalian anti-IL-12 antibody that binds to thesame region of a IL-12 protein as an antibody comprising at least oneheavy chain or light chain CDR having the amino acid sequence of atleast one of SEQ ID NOS: 1, 2, 3, 4, 5, or 6, with, or to, said cell,tissue, organ or animal.
 73. A method according to claim 72, whereinsaid effective amount is 0.001-50 mg/kilogram of said cells, tissue,organ or animal.
 74. A method according to claim 72, wherein saidcontacting or said administrating is by at least one mode selected fromparenteral, subcutaneous, intramuscular, intravenous, intrarticular,intrabronchial, intraabdominal, intracapsular, intracartilaginous,intracavitary, intracelial, intracelebellar, intracerebroventricular,intracolic, intracervical, intragastric, intrahepatic, intramyocardial,intraosteal, intrapelvic, intrapericardiac, intraperitoneal,intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal,intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine,intravesical, bolus, vaginal, rectal, buccal, sublingual, intranasal, ortransdermal.
 75. A method according to claim 72, further comprisingadministering, prior, concurrently or after said (a) contacting oradministering, at least one composition comprising an effective amountof at least one compound or protein selected from at least one of adetectable label or reporter, a TNF antagonist, an antirheumatic, amuscle relaxant, a narcotic, a non-steroid anti-inflammatory drug(NSAID), an analgesic, an anesthetic, a sedative, a local anethetic, aneuromuscular blocker, an antimicrobial, an antipsoriatic, acorticosteriod, an anabolic steroid, an erythropoietin, an immunization,an immunoglobulin, an immunosuppressive, a growth hormone, a hormonereplacement drug, a radiopharmaceutical, an antidepressant, anantipsychotic, a stimulant, an asthma medication, a beta agonist, aninhaled steroid, an epinephrine or analog, a cytokine, or a cytokineantagonist.
 76. A method according to claim 72, wherein said IL-12related condition is psoriasis.
 77. A method according to claim 72,wherein said IL-12 related condition is multiple sclerosis.
 78. Amedical device, comprising at least one isolated mammalian anti-IL-12antibody that binds to the same region of a IL-12 protein as an antibodycomprising at least one heavy chain or light chain CDR having the aminoacid sequence of at least one of SEQ ID NOS: 1, 2, 3, 4, 5, or 6,wherein said device is suitable to contacting or administerting said atleast one anti-IL-12 antibody by at least one mode selected fromparenteral, subcutaneous, intramuscular, intravenous, intrarticular,intrabronchial, intraabdominal, intracapsular, intracartilaginous,intracavitary, intracelial, intracelebellar, intracerebroventricular,intracolic, intracervical, intragastric, intrahepatic, intramyocardial,intraosteal, intrapelvic, intrapericardiac, intraperitoneal,intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal,intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine,intravesical, bolus, vaginal, rectal, buccal, sublingual, intranasal, ortransdermal.
 79. A method for producing at least one isolated mammaliananti-IL-12 antibody that binds to the same region of a IL-12 protein asan antibody comprising at least one heavy chain or light chain CDRhaving the amino acid sequence of at least one of SEQ ID NOS: 1, 2, 3,4, 5, or 6, comprising providing a host cell or transgenic animal ortransgenic plant or plant cell capable of expressing in recoverableamounts said antibody.
 80. At least one anti-IL-12 antibody produced bya method according to claim
 79. 81. At least one isolated mammaliananti-IL-12 antibody, comprising at least one human CDR, wherein saidantibody specifically binds at least one epitope comprising at least1-3, to the entire amino acid sequence of SEQ ID NO:
 9. 82. An IL-12antibody according to claim 81, wherein said antibody binds IL-12 withan affinity of at least one selected from at least 10⁻⁹ M, at least10⁻¹⁰ M, at least 10⁻¹¹ M, or at least 10⁻¹² M.
 83. An IL-12 antibodyaccording to claim 81, wherein said antibody substantially neutralizesat least one activity of at least one IL-12 protein.
 84. An isolatednucleic acid encoding at least one isolated mammalian anti-IL-12antibody having at least one human CDR, wherein said antibodyspecifically binds at least one epitope comprising at least 1-3, to theentire amino acid sequence of SEQ ID NO:
 9. 85. An isolated nucleic acidvector comprising an isolated nucleic acid according to claim
 84. 86. Aprokaryotic or eukaryotic host cell comprising an isolated nucleic acidaccording to claim
 85. 87. A host cell according to claim 86, whereinsaid host cell is at least one selected from COS-1, COS-7, HEK293,BHK21, CHO, BSC-1, Hep G2, 653, SP2/0, 293, HeLa, myeloma, or lymphomacells, or any derivative, immortalized or transformed cell thereof. 88.A method for producing at least one anti-IL-12 antibody, comprisingtranslating a nucleic acid according to claim 84 under conditions invitro, in vivo or in situ, such that the IL-12 antibody is expressed indetectable or recoverable amounts.
 89. A composition comprising at leastone isolated mammalian anti-IL-12 antibody having at least one humanCDR, wherein said antibody specifically binds at least one epitopecomprising at least 1-3, to the entire amino acid sequence of SEQ ID NO:9, and at least one pharmaceutically acceptable carrier or diluent. 90.A composition according to claim 89, further comprising at least onecomposition comprising an effective amount of at least one compound orprotein selected from at least one of a detectable label or reporter, adetectable label or reporter, a TNF antagonist, an antirheumatic, amuscle relaxant, a narcotic, a non-steroid anti-inflammatory drug(NSAID), an analgesic, an anesthetic, a sedative, a local anethetic, aneuromuscular blocker, an antimicrobial, an antipsoriatic, acorticosteriod, an anabolic steroid, an erythropoietin, an immunization,an immunoglobulin, an immunosuppressive, a growth hormone, a hormonereplacement drug, a radiopharmaceutical, an antidepressant, anantipsychotic, a stimulant, an asthma medication, a beta agonist, aninhaled steroid, an epinephrine or analog, a cytokine, or a cytokineantagonist.
 91. An anti-idiotype antibody or fragment that specificallybinds at least one isolated mammalian anti-IL-12 antibody having atleast one human CDR, wherein said antibody specifically binds at leastone epitope comprising at least 1-3, to the entire amino acid sequenceof SEQ ID NO:
 9. 92. A method for diagnosing or treating a IL-12 relatedcondition in a cell, tissue, organ or animal, comprising (a) contactingor administering a composition comprising an effective amount of atleast one isolated mammalian anti-IL-12 antibody having at least onehuman CDR, wherein said antibody specifically binds at least one epitopecomprising at least 1-3, to the entire amino acid sequence of SEQ ID NO:9, with, or to, said cell, tissue, organ or animal.
 93. A methodaccording to claim 92, wherein said effective amount is 0.001-50mg/kilogram of said cells, tissue, organ or animal.
 94. A methodaccording to claim 92, wherein said contacting or said administrating isby at least one mode selected from parenteral, subcutaneous,intramuscular, intravenous, intrarticular, intrabronchial,intraabdominal, intracapsular, intracartilaginous, intracavitary,intracelial, intracelebellar, intracerebroventricular, intracolic,intracervical, intragastric, intrahepatic, intramyocardial, intraosteal,intrapelvic, intrapericardiac, intraperitoneal, intrapleural,intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal,intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical,bolus, vaginal, rectal, buccal, sublingual, intranasal, or transdermal.95. A method according to claim 92, further comprising administering,prior, concurrently or after said (a) contacting or administering, atleast one composition comprising an effective amount of at least onecompound or protein selected from at least one of a detectable label orreporter, a TNF antagonist, an antirheumatic, a muscle relaxant, anarcotic, a non-steroid anti-inflammatory drug (NSAID), an analgesic, ananesthetic, a sedative, a local anethetic, a neuromuscular blocker, anantimicrobial, an antipsoriatic, a corticosteriod, an anabolic steroid,an erythropoietin, an immunization, an immunoglobulin, animmunosuppressive, a growth hormone, a hormone replacement drug, aradiopharmaceutical, an antidepressant, an antipsychotic, a stimulant,an asthma medication, a beta agonist, an inhaled steroid, an epinephrineor analog, a cytokine, or a cytokine antagonist.
 96. A method accordingto claim 92, wherein said IL-12 related condition is psoriasis.
 97. Amethod according to claim 92, wherein said IL-12 related condition ismultiple sclerosis.
 98. A medical device, comprising at least oneisolated mammalian anti-IL-12 antibody having at least one human CDR,wherein said antibody specifically binds at least one epitope comprisingat least 1-3, to the entire amino acid sequence of SEQ ID NO: 9, whereinsaid device is suitable to contacting or administerting said at leastone anti-IL-12 antibody by at least one mode selected from parenteral,subcutaneous, intramuscular, intravenous, intrarticular, intrabronchial,intraabdominal, intracapsular, intracartilaginous, intracavitary,intracelial, intracelebellar, intracerebroventricular, intracolic,intracervical, intragastric, intrahepatic, intramyocardial, intraosteal,intrapelvic, intrapericardiac, intraperitoneal, intrapleural,intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal,intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical,bolus, vaginal, rectal, buccal, sublingual, intranasal, or transdermal.99. A method for producing at least one isolated mammalian anti-IL-12antibody having at least one human CDR, wherein said antibodyspecifically binds at least one epitope comprising at least 1-3, to theentire amino acid sequence of SEQ ID NO: 9, comprising providing a hostcell or transgenic animal or transgenic plant or plant cell capable ofexpressing in recoverable amounts said antibody.
 100. At least oneanti-IL-12 antibody produced by a method according to claim
 99. 101. Anyinvention described herein.